UC-NRLF 


LIBRARY 

HWZVERSITY  Op  CALIFORNI4 


MICROBIOLOGY 


MARSHALL 


MICROBIOLOGY 

A  TEXT-BOOK  OF 

MICROORGANISMS  GENERAL  AND  APPLIED 

CONTRIBUTORS 

F.  T.  Bioletti.  Berkeley,  California.  J.  G.  Lipman,  New  Brunswick,  New  Jersey. 

R.  E.  Buchanan,  Ames,  Iowa.  W.  J.  MacNeal,  New  York,  New  York. 

W.  V.  Cruess,  Berkeley,  California.  E.  F.  McCampbell,  Columbus,  Ohio. 

M.  Dorset,  Washington,  D.  C.  E.  B.  Phelps,  Washington,  D.  C. 

S.  F.  Edwards,  Lansing,  Michigan.  O.  Rahn,  Elbing,  Germany. 

E.  Fidlar,  London,  Ontario.  L.  F.  Rettger,  New  Haven,  Connecticut. 
W.  D.  Frost,  Madison,  Wisconsin*  M.  H.  Reynolds,  University  Farm,  St.  Paul, 
A.  Guilliermond,  Lyons,  France.  Minnesota. 

F.  C.  Harrison,  Macdonald  College,  Que.,  Canada.    W.  G.  Sackett,  Fort  Collins,  Colorado. 
E.  G.  Hastings,  Madison,  Wisconsin.  W.  A    Stocking,  Ithaca,  New  York. 
H.  W.  Hill,  London,  Ontario.  C.  Thorn,  Washington,  D.  C. 

Arao  Itano,  Amherst,  Massachusetts.  J.  L.  Todd,  Montreal,  Quebec. 

W.  E.  King,  St.  Paul,  Minnesota.  Z.  Northrup  Wyant,  East  Lansing,  Michigan. 

EDITED  BY 

CHARLES  E.  MARSHALL 

Amherst,  Massachusetts 

PROFESSOR  OF   MICROBIOLOGY  AND   DIRECTOR  OF  GRADUATE  SCHOOL 
MASSACHUSETTS  AGRICULTURAL  COLLEGE 


THIRD  EDITION  REVISED  AND  ENLARGED 
WITH  200  ILLUSTRATIONS 


PHILADELPHIA 

P.  BLAKISTON'S  SON  &  CO 

1012  WALNUT  STREET 

LIBRARY 

UNIVERSITY  OF  CALIFORNIA 


COPYRIGHT,  1921,  BY  P.  BLAKISTON'S  SON  &  Co. 


THK  MAI'LE  TRESS  YORK  FA 


CONTRIBUTORS 


BIOLETTI,  FREDERIC  T.,  M.  S. 

Professor  of  Viticulture  and  Enology,  Viticulturist  of  Experiment  Station, 

University  of  California,  Berkeley. 
BUCHANAN,  R.  E.,  B.  S.,  M.  S.,  PH.  D. 

Professor  of  Bacteriology,  Bacteriologist  of  Experiment  Station,  and  Dean 

of  the  Graduate  College,  Iowa  State  College,  Ames. 
CRUESS,  W.  V.. 

Assistant   Professor  of   Fruit   Products,    Agricultural  Experiment   Station, 

University  of  California,  Berkeley. 
DORSET,  M.,  B.  S.,  M.  D. 

Chief  of  Biochemic  Division,  U.  S.  Bureau  of  Animal  Industry,  Washington, 

D.  C. 
EDWARDS,  S.  F.,  B.  S.,  M.  S. 

Formerly  Professor  of  Bacteriology,  Ontario  Agricultural  College,  Guelph, 

Canada.     Director  of  The  Edwards  Laboratories,  Lansing,  Michigan. 
FIDLAR,  EDWARD,  B.  A.,  M.  B. 

Formerly  Chief  of    Division  of  Pathology,   Institute    of    Public    Health; 

Pathologist   of  London  Asylum  and    of    Victoria    Hospital;    Professor  of 

Pathology,  W.  U.  Medical    Faculty;    Bacteriologist   of    London  Board  of 

Health,  London,  Ontario.    Captain,  C.  A.  M.  C. 
FROST,  W.  D.,  PH.  D.,  D.  P.  H. 

Professor  of  Agricultural  Bacteriology,  University  of  Wisconsin,  Madison. 

GUILLIERMOND,    A.,    DOCTEUR    fes    SCIENCES. 

Professor  of  Botany,  University  of  Lyon,  France. 
HARRISON,  F.  C.,  D.  Sc.,  F.  R.  S.  C. 

Principal  and  Professor  of  Bacteriology,  Macdonald  College  (Faculty  of  Agri- 
culture, McGill  University),  Macdonald  College,  Que.,  Canada. 
HASTINGS,  E.  G.,  M.  S. 

Professor  of  Agricultural  Bacteriology,  Bacteriologist  of  Experiment  Station, 

University  of  Wisconsin,  Madison. 
HILL,  H.  W.,  M.  B.,  M.  D.,  D.  P.  H. 

Formerly  Executive  Secretary,  Minnesota  Public   Health  Association,  St. 

Paul;    Director   of   Institute    of    Public    Health   of    Western    University, 

London,  Ontario,  Canada. 
ITANO,  ARAO,  B.  S.,  PH.  D. 

Associate  Professor  of  Microbiology,  Massachusetts  Agricultural  College, 

Amherst. 


VI  CONTRIBUTORS 

KING,  WALTER  E.,  M.  A.,  M.  D. 

Formerly  Professor  of  Bacteriology  and  Bacteriologist  of  Experiment  Station, 

Kansas  Agricultural  College,  Manhattan;  Assistant  Director  of  Research 

Laboratory,  Parke,  Davis  &  Co.,  Detroit,  Michigan.  Laboratory  Director, 

Beebe  Laboratories,  Inc.,  St.  Paul,  Minnesota. 
LIPMAN,  JACOB  G.,  PH.  D. 

Dean  of  Agriculture,  Rutgers  College;  Director  of  Experiment  Station,  New 

Brunswick,  New  Jersey. 
MACNEAL,  WARD  J.,  PH.  D.,  M.  D. 

Professor  of  Bacteriology  and  Director  of  the  Laboratories,  New  York  Post- 

Graduate  Medical  School  and  Hospital,  New  York. 
MCCAMPBELL,  EUGENE  F.,  PH.  D.,  M.  D. 

Professor  of  Preventive  Medicine,  Dean  of  the  Medical  College,  Ohio  State 

University. 
PHELPS,  EARLE  B.,  B.  S. 

Professor  of  Chemistry,  Hygienic  Laboratory,  U.  S.  Public  Health  Service, 

Washington,  D.  C. 
RAHN,  OTTO,  PH.  D. 

Foimerly  Assistant  Professor  of  Bacteriology,  Illinois  University,  Urbana. 

Now  Elbing,  Germany. 
RETTGER,  L.  F.,  PH.  D. 

Professor    of    Bacteriology    and  Hygiene   (in  Sheffield  Scientific   School), 

Yale  University,  New  Haven,  Connecticut. 
REYNOLDS,  M.  H.,  B.  S.,  M.  D.,  D.  V.  M. 

Professor  of  Veterinary  Medicine  and  Surgery,  Agricultural  College,  Univer- 
sity of  Minnesota;  Experiment  Station,  University  Farm,  St.  Paul. 
SACKETT,  WALTER  G.,  B.  S.,  Ph.  D. 

Bacteriologist,  Colorado  Experiment  Station,  Colorado  Agricultural  College, 

Fort  Collins. 
STOCKING,  W.  A.,  M.  S.  A. 

Professor  of  Dairy  Industiy,  Cornell  University,  Ithaca,  New  York;  Dairy 

Bacteriologist  of  the  Experiment  Station. 
THOM,  CHARLES,  PH.  D. 

Mycologist,  Bureau  of  Chemistry,  U.  S.  Department  of  Agriculture,  Wash- 
ington, D.  C. 
TODD,  J.  L.,  B.  A.,  M.  D.,  D.  Sc. 

Associate  Professor  of  Parasitology,  McGill  University,  Montreal. 
WYANT,  ZAE  NORTHRUP,  M.  S. 

Research   Associate   in    Bacteriology,   Michigan    Agricultural   Experiment 

Station,  East  Lansing. 


INTRODUCTION  TO  THE  THIRD  EDITION 


The  kindly  reception  of  Microbiology,  which  has  been  progressive, 
makes  a  revision  a  pleasurable  task. 

There  has  been  little  need  of  change  in  the  basic  facts  presented, 
but  there  is  always  room  for  a  clarification  of  thought  and  improvement 
in  arrangement.  As  time  has  passed  it  has  been  found  desirable,  also, 
to  emphasize  and  extend  some  of  the  chapters. 

Teaching  has  demonstrated  that,  in  most  instances,  the  chapters 
dealing  with  biological  products  follow  more  naturally  and  logically 
the  chapter  on  immunity.  Since  the  chapters  on  diseases  are  more  of  a 
reference  character,  they  have  been  placed  at  the  end. 

The  war  has  made  more  prominent  food  contamination,  preservation 
and  decomposition.  For  this  reason  all  chapters  considering  food  have 
been  brought  together  in  a  single  division  and  greater  attention  has 
been  given  the  subject  by  rewriting,  insertions  and  enlarging  the  scope. 
Dairy  microbiology  has  not  been  included  in  the  division  of  food  be- 
cause it  has  such  a  distinctive  field  of  its  own. 

The  editor  has  a  deep  feeling  of  indebtedness  to  the  contributors  who 
have  been  so  kindly  disposed,  ready  and  helpful  in  this  revision,  and 
to  Miss  Marion  F.  Dondale,  for  her  immeasurable  assistance. 

CHARLES  E.  MARSHALL,  EDITOR. 

.\\MIERST,  MASSACHUSETTS. 


vn 


INTRODUCTION  TO  THE  SECOND 
EDITION 


The  continued  and  growing  demand  for  "Microbiology"  has  caused 
the  contributors  to  undertake  a  thorough  revision.  In  this  they  have 
been  guided  by  the  recent  developments  in  this  branch  of  science, 
and  also  by  a  desire  to  adjust  and  rearrange  in  the  light  of  constructive 
suggestions  and  criticisms. 

The  primary  purpose  of  this  text-book  is  to  place  in  the  hands  of 
college  students  an  elementary  technical  treatise  of  the  subject  matter 
included.  No  effort  has  been  made  to  review  or  cite  literature,  for  to 
do  either  would  expand  the  volume  beyond  useful  limits.  To  provide 
an  introductory  text-book  mainly  for  recitations,  or  for  a  supplement 
to  lecture  or  laboratory  courses,  is  about  all  that  can  be  satisfactorily 
comprehended  in  a  single  project. 

The  cytological  aspect  of  microbiology  has  seemed  to  us  to  deserve 
some  emphasis,  for  it  has  become  quite  definite  and  has  been  suggest- 
ively indicating  much  of  real  value  in  connection  with  the  active  life 
processes  of  the  cell  and  microbic  activities  in  agriculture,  medicine 
and  wherever  microbiology  is  applicable. 

The  significance  of  "Intestinal  Microbiology"  has  required  a  short 
chapter  for  its  proper  presentation. 

It  has  also  been  found  desirable  to  treat  the  microbial  diseases  of 
insects,  a  growing  subject,  in  a  distinct  chapter. 

The  study  of  microorganisms  flounders  in  a  fog  of  unsettled  ideas 
for  a  proper  designation.  Whether  it  should  be  called  Protistology, 
Microbiology,  Bacteriology,  Mycology,  or  something  else  must  be  left 
for  the  future  to  determine. 

CHARLES  E.  MARSHALL,  EDITOR. 
AMHERST,  MASSACHUSETTS. 

ix 


INTRODUCTION  TO  THE  FIRST  EDITION 


By  a  process  of  adaptation  and  growth,  the  branch  of  science  com- 
monly recognized  as  "Bacteriology"  has  for  many  years  included, 
besides  the  bacterial  forms,  those  microorganisms  yielding  to  the  same 
laboratory  methods  of  study  and  investigation.  This  is  a  policy  or 
purpose  instituted  by  Pasteur.  It  is  also  the  result  of  investigations 
and  added  knowledge,  more  definite  arrangements  of  available  facts, 
and  the  highly  specialized  training  required  for  the  work.  In  short, 
technic  together  with  the  economic  relations  of  the  subject-matter 
has  no  little  influence  in  placing  limitations.  In  the  light  of  such  cir- 
cumstances, it  appears  more  pertinent  to  designate  this  text-book 
as  "Microbiology,"  perhaps  not  the  best  term,  but  one  much  in  accord 
with  French  usage. 

Agriculture,  Domestic  Science  and  certain  other  courses  in  scientific 
schools  and  colleges  call  for  the  treatment  of  the  subject  in  such  a  man- 
ner as  to  make  it  basic  to  the  interpretation  of  such  subjects  as  air 
impurities,  water  supplies,  sewage  disposal,  soils,  dairying,  fermenta- 
tion industries,  food  preservation  and  decomposition,  manufacture 
of  biological  products,  transmission  of  disease,  susceptibility  and  im- 
munity, sanitation,  and  control  of  infectious  or  contagious  diseases. 
A  strong  effort  has  been  made  to  provide  the  fundamental  and  guiding 
principles  of  the  subject  and  to  show  just  how  these  principles  fit  into 
the  subjects  of  a  more  or  less  strictly  professional  or  practical  nature. 
Here  the  instructional  work  of  the  microbiologist  stops  in  most  educa- 
tional institutions  and  the  instruction  of  the  practical  or  professional 
man  begins. 

Because  of  the  extreme  massiveness  and  diversity  of  the  subjects, 
Agriculture  and  Domestic  Science  and  Industrial  Vocations  in  general, 
a  comprehensive  consideration  of  the  subject  is  demanded.  Elimina- 
tion of  many  features  not  only  becomes  difficult  but  really  precarious, 
because  so  many  avenues  are  open  to  the  student  that  pertinency  cannot 
always  be  foreseen  or  determined.  It  is  well  to  remember,  too,  that 

xi 


Xll  INTRODUCTION    TO    THE    FIRST   EDITION 

such  aggregate  subjects  as  Agriculture  and  Domestic  Science,  unlike 
Engineering  and  Medicine,  because  of  their  youth,  have  not  developed 
to  that  stage  in  their  educational  history  where  practice  and  the  science 
upon  which  practice  should  be  founded  are  amalgamated.  The  practi- 
cal man  in  Agriculture,  and  Applied  Sciences  generally,  too  frequently 
is  so  extremely  traditional  in  his  practice  that  he  utterly  fails  to  separate 
the  true  from  the  false,  or,  in  other  words,  does  not  exercise  his  dis- 
criminative powers  at  all,  but  depends  entirely  upon  so-called  haphazard 
methods  and  self-willed  processes.  This  factor  operates  against  the 
proper  development  and  logical  study  of  any  branch  of  science  in  its 
relation  to  the  farmer,  or  manufacturer. 

The  plan  of  a  text-book  in  Microbiology  which  seeks  to  furnish 
basic  principles,  to  train  the  mind  in  logical  development  and  adjust- 
ment, and  to  prepare  the  student  to  undertake  an  intelligent  study  of 
strictly  professional  or  practical  subjects,  must  assume  a  definite  and 
systematic  arrangement.  With  this  in  mind,  the  text  has  been  divided 
into  three  distinct  parts:  Morphological  and  Cultural,  or  that  which 
deals  with  forms  and  methods  of  handling;  Physiological,  or  that  which 
deals  strictly  with  functions,  the  key  to  the  applied;  Applied,  or  that 
which  reaches  into  the  application  of  the  facts  developed  to  the  problems 
met  in  the  study  of  professional  or  practical  affairs. 

In  a  text-book,  the  product  of  several  hands,  there  is  the  most  serious 
difficulty  in  obtaining  unity  of  thought  and  expression  without  repeti- 
tion; besides,  that  very  conspicuous  weakness  of  emphasizing  some  fea- 
tures unduly  while  other  features  of  importance  are  scarcely  mentioned, 
confronts  us.  A  most  earnest  attempt  has  been  made  to  overcome 
these  faults  as  far  as  possible,  but  a  complete  mastery  of  them  cannot 
be  expected  in  the  first  product.  However,  what  is  lacked  in  unity 
and  continuity  of  expression  and  in  balance  we  sincerely  hope  will  be 
made  up,  in  part  at  least,  by  the  selection  and  the  value  of  the  material 
contributed. 

Laboratory  features  of  microbiology  have  been  eliminated  wher- 
ever it  has  been  practicable.  Should  any  demonstrations  be  added 
or  needed,  we  have  felt  that  they  may  be  easily  supplied  by  the  instruc- 
tor, who,  of  course,  will  be  governed  by  local  facilities  and  conditions. 
Although  no  space  has  been  given  to  laboratory  exercises,  it  should  not 
be  gathered  that  the  authors  of  this  book  are  any  the  less  earnest  in 
urging  a  well-organized  laboratory  course  to  supplement- the  general 


INTRODUCTION   TO    THE    FIRST   EDITION  Xlll 

instruction  as  an  essential  factor  to  a  working  appreciation  of  the 
subject. 

In  matters  of  spelling,  new  words,  and  phrases,  conservatism  has 
controlled.  Arbitrary  decisions  and  selections  have  been  forced  in 
several  instances  to  secure  clearness,  consistency  and  definiteness. 
It  is  painfully  evident  to  anyone  attempting  to  bring  system  out  of 
the  confusion  and  chaos  existing  in  many  fields  of  microbiological 
action  that  some  rearrangement  ought  to  be  undertaken.  As  usual, 
however,  this  will  be  very  slow  on  account  of  the  many  almost  insur- 
mountable difficulties. 

We  need  and  invite  helpful  suggestions  and  criticisms  at  all  times, 
for  a  valuable  text-book  of  the  nature  of  this  is  one  of  slow  growth  and 
development  and  not  of  "sport  evolution."  The  editor  is  certain  that 
each  contributor  will  welcome  suggestions  and,  further,  will  be  in  far 
better  position  to  judge  his  own  contribution  after  the  material  appears 
in  book  form  and  has  been  submitted  to  students  for  which  it  is  designed. 

No  one  better  than  the  editor  realizes  fully  the  sympathetic  part 
played  by  the  contributors.  If  any  merit  attaches  to  this  book  as  it 
finds  its  place  in  microbiological  instruction,  such  merit  should  be 
recognized  as  due  the  contributors  whose  unselfish  aims  have  made  it 
possible. 

CHARLES  E.  MARSHALL,  EDITOR. 
AMHERST,  MASSACHUSETTS. 


CONTENTS 


TITLE  PAGE iii 

CONTRIBUTORS • v 

INTRODUCTIONS  (Editor) vii 

CONTENTS  (Editor) xv 

HISTORICAL  REVIEW  (Harrison) i 

PART  I.— MORPHOLOGY  AND  CULTURE  OF  MICROORGANISMS 

GENERAL  (Editor).    OUTLINE  OF  PLANT  GROUPS  (Thorn) 
OUTLINE  OF  PROTOZOAL  GROUPS  (Todd) 

CHAPTER  I. — ELEMENTS  OF  MICROBIAL  CYTOLOGY  (Guilliermond) 15 

Cells  and  energids. — Structure  of  the  cell, — Nuclear  structure  (general  structure  of 
the  nucleus,  centriole,  value  of  the  nucleus,  forms  of  nuclei,  theory  of  binuclearity), 
cytoplasm  (appearance  of  protoplasm,  chondriosomes,  vacuoles,  reserve  products), 
membrane,  locomotion. — Reproduction, — Various  processes,  nuclear  division  (mito- 
sis, amitosis),  sexual  changes. 

CHAPTER  II. — MOLDS  (Thorn)  CYTOLOGY  (Guilliermond) 36 

Fungi  in  general, — Bacteria.  Phycomycetes,  Ascomycetes,  Basidiomycetes,  Imper- 
fect fungi. — Cytology  of  molds, — General  structure  of  molds,  cytoplasm,  nuclei, 
metachromatic  corpuscles  and  reserve  products,  cell  wall. — Molds, — Cosmopolitan 
saprophytes,  molds  of  fermentation,  parasitic  molds. — Consideration  of  groups, — 
Mucor,  Thamnidium,  Penicillium,  Aspergillus,  Monascus,  Cladosporium,  Alter- 
naria  and  Pusarium,  Oidium,  Monilia,  Dematium,  Saprolegftiaceae. 

CHAPTER  III.— YEASTS  (Bioletti)   CYTOLOGY  (Guilliermond) 61 

Morphology  of  certain  types, — Definition  and  bases  of  classification. — Cytology, — 
General  structure  of  yeasts,  cytological  phenomena  during  multiplication,  variation 
in  the  cellular  structure  during  development,  cytological  phenomena  of  the  sporula- 
tion  and  germination  of  ascospores. — The  principal  yeasts  of  importance  to  fermenta- 
tion industries, — True  yeasts,  pseudo-yeasts. — Culture  of  yeasts. 

CHAPTER  IV.— BACTERIA  (Frost)  CYTOLOGY  (Guilliermond) 79 

Forms  of  lower  bacteria, — Fundamental  form  types,  gradations,  involution  forms. 
— Size. — Motility, — Brownian  movement,  vital  movement,  organs  of  locomotion, 
character  of  movement,  rate. — Reproduction, — Vegetative  multiplication,  spore 
formation. — Cell  grouping, — Cell  aggregates  among  the  micrococci,  the  bacilli,  the 
spirilla,  Zooglcea. — Cytology  of  bacteria, — General  consideration  of  cytoplasm  and 
nucleus,  minute  consideration  of  cytoplasm  and  nucleus,  life  cycle  of  bacteria 
(Editor),  reserve  products,  general  structure  of  cell  wall,  minute  structure  of  cell  wall, 
capsules,  general  consideration  of  flagella,  minute  consideration  of  flagella. — Higher 
bacteria, — The  larger  spirochaetes,  trichobacteria,  the  sulphur  bacteria. — Classi- 
fication.— Relationship  of  bacteria. — Cultivation  of  bacteria. 

CHAPTER  V. — FILTRABLE  MICROSRGANISMS  (Dorset) 119 

A  brief  general  discussion  of  the  available  knowledge  of  filtrable  microorganisms. 

XV 


XVI  CONTENTS 

CHAPTER  VL— PROTOZOA  (Todd) 123 

Introduction. — Structure  of  protozoa. — Activities  of  protozoa, — Locomotion,  re- 
production, developmental  cycle,  encystment. — Parasitism.- — Discussion  of  classifi- 
cation.— Technic. 

PART  II.— PHYSIOLOGY  OF  MICROORGANISMS 

DIVISION  I 

INTRODUCTION  ... 145 

CHAPTER  I. — UNIT  OF  BIOLOGICAL  ACTIVITY  (Marshall  and  Itano) 147 

The  mechanism  of  cells. 

CHAPTER  II. — A  STUDY  OF    PHYSICAL  FORCES  INVOLVED   IN  BIOLOGICAL  ACTIVITIES 

(Marshall  and  Itano) 155 

Introduction, — Energy. — Solutions. — Electrical  conductivity,  ionization  and 
dissociation, — "True  reaction,"  theory  of  H  ion  concentration. — Surface  tension. — 
Adsorption. — Brownian  motion. — Diffusion,  osmosis,  dialysis,  permeability. — 
Colloids  and  crystalloids. 

CHAPTER  III. — CHEMICAL  STUDIES  OF  THE  CONTENTS  OF  MICROBIAL  CELLS  (Marshall 

and  Itano) 186 

Analyses, — Moisture,  proteins  and  other  nitrogenous  substances,  carbohydrates, 
fats,  ash  elements,  enzymes,  toxins,  vitamines. 

DIVISION  II. — NUTRITION  AND  METABOLISM  (Rahn) 

INTRODUCTION — (Revised  by  Marshall;  a  few  paragraphs  on  protozoal  nutrition  by 
Todd) : 195 

CHAPTER  I. — ENERGY  REQUIREMENTS  IN  CELLULAR  NUTRITION 199 

CHAPTER  II. — MECHANISM  OF  METABOLISM 203 

General  theory  of  metabolism, — Metabolism,  katabolism,  anabolism. — Intra-  and 
extra-cellular  fermentation. — Decomposition  of  insoluble  food,  properties  of  en- 
zymes, enzymes  of  fermentation, — Classification  of  enzymes. — Hydrolytic  enzymes, 
enzymes  of  carbohydrates,  enzymes  of  fats,  enzymes  of  proteins,  coagulating  en- 
zymes.— Zymases. — Oxidizing  enzymes. — Reducing  enzymes. — Enzymic  theory 
of  katabolism. — Enzymic  theory  of  anabolism. — General  enzymic  considerations. 

CHAPTER  III.— FOOD  OF  MICROORGANISMS 221 

Moisture  requirement. — Amount  of  food  required. — Food  for  growth, — Sources  of 
carbon,  nitrogen,  hydrogen,  oxygen,  minerals. — Food  for  energy  (oxygen  relations). 

CHAPTER    IV. — PRODUCTS  OF  MICROBIAL  ACTIVITIES 230 

General  considerations. — The  chemical  equations  of  fermentations. — Products 
from  nitrogen-free  compounds, — Sugars,  starch,  cellulose,  acids,  alcohols,  fats. — 
Products  from  nitrogenous  compounds, — Protein  bodies,  ptomaines,  urea,  uric 
acid,  hippuric  acid. — Products  from  mineral  compounds. — Oxidations,  reductions. 
— Unknown  products  of  physiological  significance, — Pigments,  aromatic  sub- 
stances, enzymes,  toxins. — Physical  products  of  metabolism, — Production  of  heat, 
production  of  light. 

CHAPTER    V. — PHYSIOLOGICAL    VARIATIONS    ASSOCIATED    WITH    METABOLISM     AND 

NUTRITION 253 

Factors  influencing  the  type  of  decomposition. 

CHAPTER  VI. — NUTRITION  OF  MICROORGANISMS  AND  THE  ROTATION  OF  ELEMENTS  IN 

NATURE 258 

Carbon  cycle. — Nitrogen  cycle. — Sulphur  cycle. — Phosphorus  cycle. 


CONTENTS  XV11 

.  DIVISION  III.— PHYSICAL  INFLUENCES  (Rahn) 

CHAPTER  I. — WATER  AS  A  PHYSJCAL  FACTOR 263 

Osmotic  pressure. — Plasmolysis  (salt  and  sugar  solutions,  colloidal  solutions). — 
Desiccation. 

CHAPTER  II. — INFLUENCE  OF  TEMPERATURE 269 

Optimum  temperature. —  Minimum  temperature. — Maximum  temperature. — 
— Biological  significance  of  the  cardinal  points  of  temperature. — End-point  of  fer- 
mentation.— Freezing. — Thermal  death-point. — Resistance  of  spores. 

CHAPTER  III. — INFLUENCE  OF  LIGHT  AND  OTHER  RAYS 278 

Phototaxis. — X-rays. — Radium  rays. 

CHAPTER  IV. — INFLUENCE  OF  ELECTRICITY •  282 

CHAPTER  V. — INFLUENCE  OF  MECHANICAL  AGENCIES 283 

Pressure. — Gravity. — Agitation. 

DIVISION  IV.— CHEMICAL  INFLUENCES  (Rahn) 

CHAPTER  I. — STIMULATION  OF  GROWTH  .    .    .    . 286 

Chemotropism  and  chemotaxis. 

CHAPTER  II.— INHIBITION  OF  GROWTH 288 

Poisons,  germicides,  disinfectants,  antiseptics,  preservatives. —  Mode  of  action. — 
Factors  influencing  disinfection. — Classification  of  disinfectants. 

DIVISION  V. — MUTUAL  INFLUENCES 

SYMBIOSIS. — METABIOSIS. — ANTIBIOSIS 297 

PART  III.— APPLIED  MICROBIOLOGY 
DIVISION  I. — MICROBIOLOGY  OF  AIR  (Buchanan) 

CHAPTER  I. — THE  MICROORGANISMS  OF  THE  AIR  AND  THEIR  DISTRIBUTION.    ...   303 
Microorganisms  present  in  the  air. — Occurrence  in  the  air. — How  microorganisms 
enter  the  air. — Conditions  for  subsidence  of  bacteria. — Determination  of  the  number 
of  bacteria  in  the  air. — Number  of  bacteria  in  the  air. — Species  of  organisms  in  the 
air. 

CHAPTER  II. — MICROBIAL  AIR  INFLUENCE  IN  FERMENTATION,  DISEASES,  ETC.     .    .    .   308 
Air  as  a  carrier  of  contagion. — Organisms  of  the  air  and  fermentation. — Freeing  air 
from  bacteria. 

DIVISION  II.— MICROBIOLOGY  OF  WATER  AND  SEWAGE 

CHAPTER  I. — MICROORGANISMS  IN  WATER   (Harrison) 310 

Classes  of  bacteria  found  in  water, — Natural  water  bacteria,  soil  bacteria  from  surface 
washings,  intestinal  bacteria  usually  of  sewage  origin. — The  number  of  bacteria  in 
rain,  snow,  hail,  etc.,  and  in  water  from  wells,  up-land,  surface  waters,  rivers,  and 
lakes. — Causes  affecting  the  increase  and  decrease  of  the  number  of  bacteria  in  water, 
— Temperature,  light,  food  supply,  oxidation,  vegetation  and  protozoa,  dilution,  sedi- 
mentation, other  causes. — Interpretation  of  the  bacteriological  analysis  of  water, — 
Quantitative  standards,  qualitative  standards. — Sedimentation,  filtration  and  purifi- 
cation of  water, — Sedimentation  and  filtration,  coagulating  basins  and  filtration, 
porous  filters,  purification  by  ozone,  purification  by  heat,  purification  by  chemicals. — 
Location  and  construction  of  wells. 


XV111  CONTENTS     . 

CHAPTER  II. — MICROBIOLOGY  OF  SEWAGE  (Phelps) 330 

Bacterial  flora  of  sewage. — Types  of  sewage  bacteria, — Putrefactive  and  anaerobic 
bacteria  (the  liquefaction  of  protein,  the  fermentation  of  cellulose,  the  saponification 
of  fats,  the  fermentation  of  urea,  the  reduction  of  sulphates  and  nitrates),  oxidizing 
bacteria  (the  production  of  nitrates  and  nitrites,  other  oxidizing  reactions),  patho- 
genic bacteria  (prevalence  and  longevity,  life  in  septic  tanks  and  filters). — The  culti- 
vation of  sewage  bacteria, — Filters,  anaerobic  tanks. — The  destruction  of  sewage 
bacteria, — By  biological  processes,  by  chemical  processes. 

DIVISION  III. — MICROBIOLOGY  OF  SOIL  (Lipman) 

CHAPTER  I. — MICROORGANISMS  AS  A  FACTOR  IN  SOIL  FERTILITY 345 

Introduction. — The  soil  as  a  culture  medium. — Moisture  relations, — The  amount 
and  distribution  of  rain  fall,  range  of  soil  moisture,  effect  of  drouth  and  excessive 
moisture. — Colloidal  nature  of  the  soil. — Aeration, — Mechanical  composition  of 
soils,  aerobic  and  anaerobic  activities,  rate  of  oxidation  of  carbon,  hydrogen  and 
nitrogen,  the  mineralization  of  organic  matter. — Temperature, — Influence  of  cli- 
mate and  season,  early  and  late  soils,  production  and  assimilation  of  plant  food. — 
Reaction. — Range  of  soil  acidity,  causes  of  soil  acidity,  soil  reaction  and  hydrogen- 
ion  concentration,  change  of  reaction  produced  by  microorganisms  in  the  medium, 
effect  of  reaction  on  number  and  species. — Food  supply, — Organic  matter,  the 
mineral  portion  of  the  soil. — Biological  factors, — Fungi,  algae,  protozoa,  higher 
plants,  bacteria  (numbers  and  distribution,  bacteria  in  productive  and  unproduc- 
tive soils,  distribution  at  different  depths,  seasonal  variations  of  bacterial  numbers 
and  activities,  morphological  and  physiological  groups). — Methods  of  study, — 
Methods  for  counting  bacteria,  quantitative  relations,  qualitative  reaction,  trans- 
formation reactions,  rate  of  oxidation  of  carbon,  rate  of  oxidation  of  nitrogen,  addi- 
tion of  nitrogen,  reactions  concerning  calcium,  magnesium,  sulphur,  phosphorus. 

CHAPTER  II. — DECOMPOSITION  OF  ORGANIC  MATTER  IN  THE  SOIL 375 

Carbohydrates, — Origin,  decomposition  of  cellulose,  the  production  of  methane  and 
hydrogen,  oxidation  of  methane,  hydrogen,  and  carbon  monoxide,  the  cleavage  and 
fermentation  of  sugars,  starches,  and  gums. — Fats  and  waxes, — Origin  and  decompo- 
sition.— Organic  acids, — Sources,  transformation  and  accumulation. — Protein 
bodies, — Amount  and  quality,  carbon-nitrogen  ratio. — Transformation  of  nitrogen 
compounds, — Ammonification,  nitrification,  denitrification. — Analytical  and  syn- 
thetical reactions, — Amount  of  bacterial  substance  in  the  soil,  availability  of  bacterial 
matter,  transformation  of  peptone,  ammonia,  and  nitrate  nitrogen. 

CHAPTER  III. — FIXATION"  OF  ATMOSPHERIC  NITROGEN.     (Methods  of  Soil  Inoculation, 

by  Edwards.) 400 

The  source  of  nitrogen  in  soils, — Early  theories,  chemical  and  biological  relations. — 
Non-symbiotic  fixation  of  nitrogen, — Historical,  anaerobic  species,  aerobic  species, 
energy  relations. — Symbiotic  fixation, — Historical,  modes  of  entrance  and  devel- 
opment, resistance,  immunity,  and  physiological  efficiency,  mechanism  of  fixation, 
variations  and  specialization,  relation  to  environment. — Soil  inoculation, — Methods 
of  soil  inoculation, — Inoculation  with  legume  earth,  inoculation  with  pure  cultures, 
etc.  (Edwards.) 

CHAPTER  IV.— CHANGES  IN  ORGANIC  CONSTITUENTS 41? 

Weathering  process, — Origin  and  formation  of  soil,  influence  of  biological  factors. — 
Lime  and  magnesia, — Removal  and  regeneration  of  carbonates,  lime  as  a  base,  effect 
of  calcium,  magnesium  compounds  upon  bacterial  activities. — Phosphorous, — Avail- 
ability of  phosphates,  relation  of  phosphorus  to  decay  and  nitrogen-fixation. — Sul- 
phur,— Sulphur  compounds  in  the  soil,  sulphur- phosphate  composts,  sulphur  bac- 
teria, sulphofication,  sulphate  reduction. — Potassium, — The  transformation  of 
potassium  compounds  in  the  soil. — Other  mineral  constituents, — Iron,  aluminum, 
manganese,  and  copper.— Antagonism. — Variability  in  soil  fertility  investigations. 


CONTENTS  XIX 


DIVISION  IV. — MICROBIOLOGY  OF  MILK  AND  MILK  PRODUCTS 

CHAPTER  I. — THE  RELATION  OF  MICROORGANISMS  TO  MILK.     (Stocking.)     (The  Acid- 
forming  Bacteria,  by  Hastings.) 428 

Character  of  milk. — Absorbed  taints  and  odors. — Changes  due  to  microorganisms. 
— Microbial  content  of  milk, — Common  milk,  special  milks,  certified  milk. — 
— Sources  of  microorganisms  in  milk, — Interior  of  cow's  udder  (healthy  udders, 
diseased  udders),  exterior  of  cow's  body,  atmosphere  of  stable  and  milk  house,  the 
milker,  utensils,  water  supply. — Methods  of  preventing  contamination  of  milk, — 
Individual  cows,  care  of  the  cow's  body,  avoid  dust  in  atmosphere,  dairy  utensils, 
the  milker. — Groups  or  types  of  microorganisms  found  in  milk,  and  their  sources, — 
General  significance  of  acid-forming  bacteria,  groups  of  acid-forming  bacteria  (char- 
acteristics of  the  Bad.  lactis  acidi  group,  characteristics  of  the  B.  coli-aerogenes 
group,  characteristics  of  the  Bact.  bulgaricus  group,  characteristics  of  the  coccus 
group)  (Hastings),  bacteria  having  no  perceptible  effect  upon  milk,  the  casein-di- 
gesting or  peptonizing  bacteria,  pathogenic  organisms. — Factors  influencing  the 
developing  of  microorganisms  in  milk, — Initial  contamination,  straining,  aera- 
tion, centrifugal  separation,  temperature,  pasteurization,  the  use  of  chemicals. — 
The  normal  development  of  microorganisms  in  milk, — -Germicidal  period,  period 
from  end  of  germicidal  action  to  time  of  curdling,  period  from  time  of  curdling  until 
acidity  is  neutralized,  final  decomposition  changes. — Abnormal  fermentations  in 
milk, — Gassy  fermentation,  sweet  curdling  fermentation,  ropy  or  slimy  fermenta- 
tion, bitter  fermentation,  alcoholic  fermentation,  other  fermentations. — The  com- 
mercial significance  of  microorganisms  in  milk, — Relation  of  dirt  contamination  to 
germ  content. — Milk  as  a  carrier  of  disease-producing  organisms, — (acid  forms, 
neutral  forms,  injurious  organisms,  epidemic  diseases,  non-epidemic  diseases). — 
Bacteriological  analysis  of  milk. — Bacteriological  milk  standards. — The  value  of 
bacteriological  milk  standards  and  analyses. 

CHAPTER  II. — THE  RELATIONS  OF  MICROORGANISMS  TO  BUTTER  (Hastings) 47  • 

Types  of  butter, — Sweet  cream  butter,  sour  cream  butter. — The  flavor  of  butter,— 
Control  of  butter  flavor,  kinds  and  numbers  of  bacteria  in  cream,  spontaneous  ripen- 
ing of  cream,  use  of  cultures  in  butter  making,  commercial  cultures,  use  of  pure  cul- 
tures in  raw  cream,  use  of  pure  cultures  in  pasteurized  cream,  pure  cultures  in  oleo- 
margarine and  renovated  butter,  abnormal  flavors  of  butter. — Decomposition 
processes  in  butter. — Pathogenic  bacteria  in  butter. 

CHAPTER  III. — RELATION  OF  MICROORGANISMS  TO  CHEESE  (Hastings) 486 

General. — Types  of  cheese, — Acid-curd  cheeses,  rennet-curd  cheeses. — Conditions  af- 
fecting the  making  of  cheese, — Quality  of  milk,  tests  for  the  quality  of  milk,  ripening 
of  milk,  curdling  of  milk,  manipulation  of  the  curd,  ripening  of  cheese  (theories  of 
cheese  ripening,  present  knowledge  of  causal  factors,  causes  of  proteolysis,  preven- 
tion of  putrefaction,  other  groups  of  bacteria  in  cheese,  flavor  production  in  cheese). 
— Abnormal  cheeses, — Gassy  cheese,  miscellaneous  abnormalities  of  cheese  (bitter 
cheese,  colored  cheese,  putrid  cheese,  moldy  cheese). — Specific  kinds  of  cheese, — 
Cheddar  cheese,  Emmenthaler  cheese,  Roquefort  cheese,  Gorgonzola  cheese,  Stilton 
cheese,  Camembert  cheese. 

CHAPTER  IV. — RELATION  OF  MICROORGANISMS  TO  SOME  SPECIAL  DAIRY  PRODUCTS 

(Stocking) 504 

General. — Condensed  milk, — Sweetened  condensed  milk,  unsweetened  condensed 
or  evaporated  milk,  concentrated  milk,  powdered  milk. — Canned  butter,  and 
cheese. — Special  milk  drinks  made  by  the  action  of  microorganisms, — Kumyss, 
kefir,  leben,  yoghurt,  artificial  buttermilk. — Ice  cream. 


XX  CONTENTS 

DIVISION  V. — MICROBIOLOGY  OF  FOODS 

CHAPTER  I. — DESICCATION,  EVAPORATION,  AND  DRYING  OF  POODS  (Buchanan)  .  .  .516 
Agencies  that  bring  about  changes  in  dried  foods. — Factors  which  inhibit  growth  of 
microorganisms  in  food. — Methods  of  drying, — Carbohydrate  foods,  as  fruits, 
macaroni,  vermicelli,  copra,  syrups,  molasses,  jellies,  jams;  fats,  as  cotton  seed, 
olive,  and  other  oils,  etc.;  protein  foods,  as  jerked  meat,  dried  beef,  dried  fish,  pem- 
mican,  beef  extract,  gelatin,  somatose,  milk,  eggs,  etc. 

CHAPTER  II. — HEAT  IN  THE  PRESERVATION  OF  FOOD  PRODUCTS  (Edwards) 524 

Historical  resume1. — Economic  importance, — From  the  standpoint  of  health  and 
dietetics,  and  from  the  standpoint  of  commerce. — Alteration  of  foods, — Physical 
changes  (appearance,  mechanical  disintegration),  chemical  changes  (appearance, 
chemical  change,  palatability  and  digestibility) ,  biological  changes  (vital  disorganiza- 
tion, normal  flora  and  fauna). — Pasteurization, — Economic  consideration,  specific 
application  (beer,  fruit  juices,  milk  and  cream,  condensed  milk). — Processing  or 
sterilization, — Economic  considerations,  specific  application  (meat,  fish,  vegetables, 
and  fruits). — Controlling  factors  in  successful  canning, — Cleanliness,  soundness  of 
raw  material,  receptacle,  water  supply,  degree  of  heat  required. — Home  canning. — 
Spoilage, — Microbiological,  detection  of  spoiled  goods. — Disposal  of  factory  refuse. 

CHAPTER  III. — THE  PRESERVATION  OF  FOOD  BY  COLD  (MacNeal) 542 

Introduction. — The  effects  of  refrigeration  upon  foods  in  general, — Changes  during 
chilling,  changes  during  storage,  changes  after  storage. — Refrigeration  of  certain 
foods, — Meat,  fish,  poultry,  eggs,  milk,  butter,  fruits  and  vegetables. — Legal  con- 
trol of  the  cold-storage  industry. 

CHAPTER  IV. — PRESERVATION  OF  FOOD  BY  CHEMICALS  (MacNeal) 550 

The  effects  of  preservatives  upon  foods  in  general, — The  process  of  curing,  the  period 
of  storage,  the  after-storage  changes. — The  chemical  preservation  of  certain  foods, — 
Meats,  fish,  dairy  products,  prepared  vegetables,  and  fruits. — The  nutritive  value 
of  preserved  foods. — The  effects  of  food  preservatives, — Substances  which  preserve 
by  their  physical  action,  substances  which  preserve  by  their  chemical  action,  inor- 
ganic food  preservatives,  organic  food  preservatives,  substances  added  to  foods  to 
improve  the  apparent  quality. — The  legal  control  of  the  preservation  of  foods  by 
chemicals. 

CHAPTER    V. — MICROBIOLOGY  OF  FERMENTED  FOODS 559 

Compressed  yeast, — yeast  as  food  (Cruess). — Bread  (Cruess). — Vegetables 
(Cruess). — Olive  pickling  and  canning  (Cruess). — Silage  (Cruess). — Malt  syrups 
(Cruess). — Tobacco  (Cruess).— Starch  (Bioletti). — Sugar  (Bioletti). — Tea  (Biol- 
etti). 

CHAPTER  VI. — MICROBIAL  FOOD  POISONING  (MacNeal)    .    . 581 

General  considerations. — Infections  of  food-producing  animals  transmissible  to 
man. — Human  infections  transmitted  in  food. — Food  poisoning  due  to  the  growth 
of  saprophytic  bacteria  in  the  food, — Poisonous  meat,  sausage,  fish,  shell  fish,  milk, 
cream,  cheese,  and  vegetable  food. — Specific  diseases  due  to  food  poisoning, — 
Botulism,  and  Bacillus  botulinus,  ergotism,  pellagra. — The  chemical  nature  of  food 
poisons. 

CHAPTER  VII. — MICROORGANISMS  OF  THE  DIGESTIVE  TRACT  (MacNeal) 593 

Introduction. — Microorganisms  of  certain  portions  of  the  alimentary  canal, — Mi- 
croorganisms of  the  mouth,  microorganisms  of  the  stomach,  microorganisms  of  the 
intestines,  microorganisms  of  the  feces. — General  method  of  study, — Collection  of 
material. 


CONTENTS  XXI 

DIVISION   VI. — MICROBIOLOGY   OF   ALCOHOLIC   FERMENTATION   AND   DERIVED 

PRODUCTS  (Bioletti) 

CHAPTER  I.— WINE 603 

Grape  juice  and  wine  as  culture  media. — The  microorganisms  found  on  grapes, — 
Molds,  yeasts,  pseudo-yeasts,  bacteria. — The  microorganisms  found  in  wine, — 
Aerobic  organisms  (mycodermae,  acetic  bacteria),  anaerobic  organisms  (slime- 
forming  bacteria,  propionic  and  lactic  bacteria,  mannitic  bacteria,  butyric  bac- 
teria).— Control  of  the  microorganisms, — Before  fermentation,  during  fermenta- 
tion, after  fermentation. — Prohibition  and  wine. 

CHAPTER  II.— BEER 622 

Raw  materials  and  microorganisms  of  brewing, — Grains  employed,  yeasts  of  beer, 
kinds  of  beer. — Process  of  brewing, — Outline,  malting  (production  of  enzymes), 
work  of  enzymes  and  bacteria,  fermentation  (work  of  yeast),  after  treatment. — 
Diseases  of  beer. 

CHAPTER  III. — MISCELLANEOUS  ALCOHOLIC  BEVERAGES  AND  PRODUCTS 628 

Cider  and  perry. — Fermented  beverages  of  various  fruits. — Hydromel  or  mead. — 
Miscellaneous  fermented  beverages, — Mexican  pulque,  sake,  pombe,  ginger  beer. — 
Distilled  alcohol, — Introduction  (uses  and  sources  of  alcohol), — Methods  (prep- 
aration of  the  sugar  solution,  fermentation). 

CHAPTER  IV. — MANUFACTURE  OF  VINEGAR 636 

Acetic  fermentation, — Nature  and  origin  of  vinegar,  vinegar  bacteria. — Processes  of 
manufacture, — Raw  materials,  fermentation,  starters  and  pure  cultures,  apparatus, 
domestic  method,  Orleans  method,  Pasteur  method,  Rapid  methods,  rotating 
barrels,  function  of  the  film,  after  treatment. — Diseases. 

.  DIVISION  VII. — MICROBIOLOGY  OF  SPECIAL  INDUSTRIES 

CHAPTER  I. — SPECIAL  INDUSTRIAL  FERMENTED  PRODUCTS 649 

Acetone  and  acetic  acid  (Cruess). — Lactic  acid  (Cruess). — Citric  acid  (Cruess). — 
White  lead  (Cruess) .--Leather  (Cruess). — Indigo  (Bioletti). — Retting  (Bioletti). 


DIVISION  VIII. — MICROBIOLOGY  OF  THE  DISEASES  OF  MAN  AND  DOMESTIC 

ANIMALS 

CHAPTER  I. — METHODS  AND  CHANNELS  OF  INFECTION  (McCampbell) 659 

Infection  defined. — Microorganisms  of  diseases  considered  and  classified, — Patho- 
genic bacteria,  pathogenic  protozoa,  ultra-microscopic  microorganisms  or  viruses, 
the  distribution  of  pathogenic  microbic  agents  in  nature. — The  occurrence  of  patho- 
genic microbic  agents  upon  and  in  the  bodies  of  healthy  animals  and  man. — The 
manner  in  which  infectious  agents  enter  the  body  and  their  sources, — Air-borne  infec- 
tions, dust  infection,  droplet  infections,  water-borne  infections,  infections  from 
soil,  infection  from  food,  animal  carriers  of  infection,  human  carriers  of  infection, 
contact  infection. — The  routes  by  which  infectious  microorganisms  enter  the  body. — 
Variation  in  infection. — The  factors  which  influence  the  results  of  an  infection, — 
Virulence,  number,  avenue,  resistance. — The  exact  cause  of  infections, — Soluble  tox- 
ins, endotoxins,  toxic  bacterial  proteins,  other  possible  exact  causes. — The  methods 
by  which  infectious  microorganisms  a're  disseminated. — The  methods  by  which  in- 
fectious microorganisms  are  eliminated  from  the  body. — The  effect  of  infectious 
microorganisms  upon  the  body, — The  period  of  incubation,  local  reactions,  general 
reactions  (metabolism,  blood-forming  organs,  parenchymatous  tissues,  epithelial  and 
endothelial  tissues,  erythrocytes  and  leucocytes,  antibody  formation). 


XX11  CONTENTS 

CHAPTER  II. — IMMUNITY  AND  SUSCEPTIBILITY  (McCampbell) 684 

General, — Definition,  hypersusceptibility  or  anaphylaxis,  predisposition  and  non- 
inheritance  of  infectious  diseases. — Immunity, — Natural  immunity  and  susceptibility 
(racial  immunity  and  susceptibility,  familial  immunity  and  susceptibility,  individual 
immunity  and  susceptibility),  factors  of  natural  immunity  (the  protection  afforded 
the  body  by  the  surfaces,  skin  and  cutaneous  orifices,  subcutaneous  tissue,  the  ex- 
posed mucous  membranes  of  the  body,  nasal  cavity,  mouth,  lungs,  stomach,  intes- 
tines, genito-urinary  tract,  conjunctiva,  the  protective  nature  of  inflammatory 
processes,  natural  antitoxins,  natural  antibacterial  substances,  normal  hemolysins, 
normal  agglutinins,  normal  precipitins) ,  acquired  immunity  (active  immunity,  pas- 
sive immunity). — The  origin  and  occurrence  of  antibodies, — Antitoxins  (the  mech- 
anism of  the  neutralization  of  toxin  by  antitoxin,  units  of  antitoxin),  lysins  and 
bactericidal  substances  (the  structure  of  lysins,  deviation  of  complement,  the  deflec- 
tion of  the  complement  as  a  test  for  antibodies),  cytotoxins  and  cytolysins,  opsonins 
and  phagocytosis  (opsonic  index,  hemoopsonins),  agglutinins  (normal  agglutinins.  the 
production  of  agglutinins,  the  distribution  of  agglutinins  in  the  blood,  inherited 
agglutinins,  the  substances  concerned  in  agglutination,  structure  of  agglutinins  and 
agglutinogens,  agglutinoids,  the  stages  of  agglutination,  hemoagglutinins),  precip- 
itins (normal  precipitins,  mechanism  of  the  formation  of  precipitins,  autoprecipitins 
and  isoprecipitins,  the  phenomena  of  specific  inhibition,  antiprecipitins,  the  precip- 
itinogen,  precipitate,  coprecipitins,  the  forensic  use  of  precipitins). — The  theories  of 
immunity, — Noxious  retention  theory,  exhaustion  theory,  Ehrlich's  side-chain 
theory,  phagocytic  theory. 

CHAPTER  III. — MANUFACTURE  OF  VACCINES  (King) 724 

Introduction. — Actively  immunizing  substances  (vaccines), — Attenuated  viruses, 
smallpox  vaccine,  blackleg  vaccine,  blackleg  aggressin,  blackleg  filtrate,  rabies 
vaccine,  Dorset-Niles  hog  cholera  serum,  anthrax  vaccine,  tuberculosis  vaccine. — 
Bacterial  vaccines  (bacterins), — Typhoid  fever,  pneumonia,  influenza-pneumonia, 
canine  distemper,  Asiatic  cholera,  bubonic  plague. — Sensitized  vaccine. — Toxin- 
antitoxin  mixture. 

CHAPTER  IV. — THE   MANUFACTURE  OF  ANTISERA  AND  OTHER  BIOLOGICAL  PRODUCTS 

RELATED  TO  SPECIFIC  INFECTIOUS  DISEASES  (King).   .    . 740 

Antitoxic  sera, — Diphtheria  antitoxin,  tetanus  antitoxin,  perfringens  antitoxin. — 
Antimicrobial  sera, — Antimeningococcic,  antistreptococcic,  '  antigonococcic,  anti- 
pneumococcic,  Dorset-Niles  (antihog  cholera),  antirabic,  antidysenteric,  preserva- 
tion of  antisera. — Tuberculins, — Koch's  old,  other  tuberculins. — Mallein. — Suspen- 
sions for  the  agglutination  tests. — Substances  used  for  diagnostic  tests, — Luetin, 
antigens,  Schick  test. 

CHAPTER  V. — CONTROL  OF  INFECTIOUS  DISEASES  (Hill) '754 

Principles. — Practice. — Public  health  methods  as  revised  and  promulgated  by  the 
Institute  of  Public  Health,  London,  Canada, — Householder's  responsibility  to 
board  of  health,  physician's  responsibility  to  board  of  health,  penalties,  definitions, 
rules  for  release  of  cases  from  isolation,  placarding  of  house,  quarantine  periods  for 
contacts,  observation  versus  quarantine,  regulations  regarding  visitors,  in  case  of 
death. — Disinfection. — Carriage  of  infection  by  biological  agents. 

CHAPTER  VI. — MICROBIAL  DISEASES  OF  MAN  AND  DOMESTIC  ANIMALS  (various  authors)  775 
Diseases  caused  by  molds  and  yeasts, — Pneumomycosis,  aspergillosis,  secondary 
infections  (Thorn),  thrush  (Thorn),  dermatomycoses,  barber's  itch,  etc.  (Thom), 
favus  (Thom),  actinomycosis  (Reynolds),  mycetoma  (Fidlar),  mycotic  lymphangitis 
(Reynolds). — Diseases  caused  by  bacteria, — Botryomycosis  (Reynolds),  gonor- 
rhcea  (Fidlar) ,  epidemic  cerebro-spinal  menirfgitis  (Fidlar) ,  infectious  mastitis  (Rey- 
nolds), Malta  fever  (Fidlar),  staphylococcic  infections  (Fidlar),  streptococcic 
infections  (Fidlar),  pneumonia  (Fidlar),  anthrax  (Harrison),  bacillary  white  diar- 
rhaea  of  young  chicks  (Rettger),  chicken  cholera  (Harrison),  chronic  bacterial  en- 
teritis (Reynolds),  'contagious  abortion  (MacNeal),  diphtheria  (Fidlar),  dysentery 


CONTENTS  XX111 

(Fidlar),  fowl  diphtheria  (Harrison),  glanders  (Reynolds),  influenza  (Fidlar),  whoop- 
ing cough  (Fidlar),  haemorrhagic  septicaemia  (Reynolds),  leprosy  (Fidlar),  plague 
(Fidlar),  swine  erysipelas  (Dorset),  tuberculosis  (Reynolds),  foot  rot  of  sheep 
(Dorset),  malignant  cedema  (Fidlar),  symptomatic  anthrax  (Reynolds),  tetanus 
(Fidlar),  typhoid  fever  (Fidlar),  Asiatic  cholera  (Fidlar). — Microbial  diseases  as  yet 
unclassified, — Scarlet  fever,  measles,  German  measles,  Duke's  disease,  smallpox, 
chickenpox,  mumps  (Hill),  canine  distemper  (Dorset),  cattle  plague  (Dorset), 
contagious  bovine  pleuro-pneumonia  (Dorset),  cowpox,  horsepox  and  sheeppox 
(King),  dengue  (Dorset),  foot-and-mouth  disease  (Dorset),  fowl  plague  (Dorset), 
hog  cholera  (Dorset),  horse  sickness  (Dorset),  infantile  paralysis  (Dorset),  pella- 
gra (MacNeal),  rabies  (MacNeal),  swamp  fever  (Reynolds),  typhus  fever  (Dorset), 
yellow  fever  (Dorset), — Diseases  caused  by  protozoa  (Todd), — Rhizopoda:  amoe- 
bic dysentery,  entero-hepatitis  of  turkeys;  flagellata  and  Leishmania:  kala-azar, 
infantile  kala-azar,  Delhi  boil;  trypanosoma:  sleeping  sickness,  human  trypano- 
somiasis  of  South  America,  trypanosomiases  of  animals;  sporozoa;  coccidia; 
coccidiosis  of  rabbits,  avian  coccidiosis;  haemosporidia:  malaria,  red  water,  East 
Coast  fever,  oroya  fever,  anaplasmosis;  sarcosporidia;  haplosporidia;  myxosporidia; 
microsporidia;  infusoria:  balantidium  enteritis;  parasites  of  uncertain  position: 
relapsing  fever,  syphilis,  yaws  or  frambcesia,  other  spirochaetal  diseases. 

DIVISION  IX.— MICROBIAL  DISEASES  OF  INSECTS  (Wyant) 

INTRODUCTION. — Bacterial  disease  of  June  Beetle  larvae,  Lachnosterna  spp. — Flacherie 
(silk  worm). — "Japanese  gipsy-moth,  disease." — Bacterial  disease  of  locusts. — Bacil- 
lary  septicaemia  of  caterpillars,  Arctia  caja. — Graphitosis. — American  foul  brood. — 
Septicaemia  of  the  cockchafer,  Melolontha  vulgaris. — European  foul  brood. — Bac- 
terial septicaemia  of  larvae  of  the  Lamellicornce. — Bacterial  disease  of  the  gut- 
epithelium  cf  the  lug-worm,  Arenicola  ecaudata. — Pseudograsserie  of  the  gipsy- 
moth  caterpillar. — Sacbrood  of  bees. — Wilt  disease  or  flacherie  of  the  gipsy-moth 
caterpillar,  Porthetria  dispar. — Pebrine. — Nosema-disease  of  bees. — Miscellaneous 
insect  diseases, — Entomophthoraceae  (Thorn), — Other  microbial  diseases  (Wyant). 
— General  pathology  and  immunity  studies 905 

DIVISION  X. — MICROBIAL  DISEASES  OF  PLANTS  (Sackett) 
INTRODUCTION 949 

CHAPTER  I.— BLIGHTS 951 

Stem  blight  of  alfalfa. — Bacteriosis  of  beans. — Blight  of  lettuce. — Blight  of  mulberry. 
— Blade  blight  of  dats. — Stem  blight  of  field  and  garden  peas. — Pear  blight. — 
Streak  disease  of  sweet  peas  and  clovers. — Tomato  blight. — Walnut  blight. 

CHAPTER  II. — GALLS  AND  TUMORS 966 

Crown  gall. — Olive  knot. — "Fingers  and  toes"  of  cabbages  (Todd). — Tuberculosis 
of  sugar  beets. 

CHAPTER  III.— LEAF  SPOTS 973 

Citrous  canker. — Angular  leaf-spot  of  cucumbers. — Leaf-spot  of  the  larkspur. — 
Bacterial  spot  of  plum  and  peach. — Disease  of  sugar  beet  and  nasturtium  leaves. 

CHAPTER  IV— ROTS 978 

Black  rot  of  cabbage. — Wakker's  hyacinth  disease. — Basal  stem  rot  of  potatoes. — 
Bud  rot  of  cocoanut. — Brown  rot  of  orchids. — Rot  of  cauliflower. — Soft  rot  of  calla 
lily. — Soft  rot  of  carrot  and  other  vegetables. — Soft  rot  of  hyacinth. — Soft  rot  of 
muskmelon. — Soft  rot  of  the  sugar  beet. 

CHAPTER  V.— WILTS 988 

Wilt  of  cucurbits. — Wilt  of  sweet  corn. — Wilt  of  tomato,  egg  plant,  Irish  potato,  and 
tobacco. — Additional  bacterial  diseases. 

INDEX  OF  CONTRIBUTORS 993 

INDEX  OF  SUBJECTS 995 


LIST  OF  ILLUSTRATIONS 


Frontispiece 

1.  Jansen's  Microscope 2 

2.  Kingdom  of  the  Protista,  diagrammatic 12 

3.  Cells  of  Saccharomyces  cerevisia 16 

4.  Cells  made  up  of  energids 16 

5.  Diffuse  nuclei  of  bacteria 17 

6.  Nuclei  in  Cyanophycea 17 

7.  Chromidia  in  protozoa  .    . 18 

8.  Micro-  and  macro-nucleus  in  an  infusorian 19 

9.  Division  of  micro-nucleus  and  chondriosomes 19 

16.  Formation  of  chloroplasts 20 

11.  Mitochondria  developing  into  amyloplasts 21 

12.  Chloroplasts  of  different  forms 21 

13.  Metachromatic  corpuscles 23 

14.  Illustrating  cyst  and  thread  membranous  walls 24 

15.  Organs  of  locomotion  in  bacteria 25 

1 6.  Division  of  Spongomonas  uvella  and  Monas  termo 26 

17.  Transverse  section  illustrating  trichocysts  and  cilia  attachments 26 

18.  Schizogony  in  Amoeba  polypodia 27 

19.  Sporogony  in  Saccharomyces  cerevisia,  B.  mycoides  and  Leucocytozoon  lovati.    27 

20.  Karyokinesis  in  Acanthocystis  aculeata  and  Coleosporium  senecionis  ....    29 

21.  Protomitosis  in  Amoeba  mucicola,  Amoeba  froschi,  Euglena  splendens,  and 

Amoeba  diplomitotica 31 

22.  Mesomitosis  in  Pelomyxa  palustris,  Urospora  lagidis,  and  Galactima  succosa.   33 

23.  Conjugation  in  Schizosaccharomyces  octosporus 34 

24.  Nuclei  in  mycelium  of  Thamnidium  elegans  and  Mucor  circinelloides.  .   .    .41 

25.  Fragments  of  mycelia  of  molds  with  dividing  nuclei 41 

26.  Filaments  of  molds  showing  chondrium 43 

27.  Nucleus  of  Mucor  in  various  stages  of  division 43 

28.  Metachromatic  corpuscles  in  Dematium 44 

29.  Metachromatic  corpuscles  in  asci 44 

30.  Metachromatic  corpuscles  in  conidia  .    .    . 45 

31.  Metachromatic  corpuscles  in  cell  of  perithecium  of  Peslularia  vesiculosa  .  .  46 

32.  Mucor,  general ". 49 

33.  Mucor,  zygospore 49 

34.  Penicillium  expansum 52 

35.  Aspergillus  glaucus 55 

36.  Aspergillus  fumigatus,  A.  nidulans - 55 

37.  Cladosporium  herbarum 57 

38.  Spores  of  Alternaria 57 

39.  Fusarium 57 

40.  Oidium  lactis 58 

41.  Monilia  Candida 59 

42.  Monilia  sitophila,  oidia  in  chains 59 

43.  Yeast  cell 62 

XXV 


XXVI  LIST    OF   ILLUSTBATIONS 

44.  Spore-bearing  yeast  cells 63 

45.  Saccharomyces  cerevisia  showing  vacuoles  and  metachromatic  corpuscles 

stained 64 

46.  Saccharomyces  cerevisia  showing  cells  with  nuclei,  nuclear  division  and 

glycogenic  vacuoles  with  grains 64 

47.  Saccharomyces  cerevisia  showing  cells  stained  by  a  special  method  re- 

vealing a  chondrium  consisting  of  granular-  and  rod-mitochondria. .    .  64 

48.  Saccharomyces  ceremsia  with  both  nucleus  and  metachromatic  granules   .  65 

49.  Saccharomyces  ellipsoideus  cells  with  nucleus 66 

50.  Copulation  and  sporulation  in  Schizosaccharomyces  octosporus 68 

51.  Various  stages  of  nuclear  division  during  sporulation  in  Schizosaccharo- 

myces octosporus 68 

52.  Cellular  fusion  in  Schizosaccharomyces  pombe .  69 

53.  Heterogamous  copulation  in  Zygosaccharomyces  chevalieri 70 

54.  Sporulation  in  Saccharomyces  ludwigii 71 

55.  Germination  of  ascospores  in  Saccharomyces  ludwigii 72 

56.  Wine  and  beer  yeasts .  74 

57.  Wild  and  pseudo-yeasts 77 

58.  Types  of  micrococci 79 

59.  Types  of  bacilli 79 

60.  Types  of  spirilla 80 

61.  Involution  forms 80 

62.  The  division  of  bacterial  cells .v 83 

63.  The  formation  of  spores 85 

64.  Location  of  spores  in  bacterial  cells 85 

65.  Spore  germination 86 

66.  Division  forms  of  micrococci 87 

67.  Division  forms  of  bacilli. 88 

68.  Threads  of  Bad.  anthracis 88 

69.  Plasmolytic  changes. 89 

70.  Karyokinetic  appearances  in  Bad.  gammari 91 

71.  B.  megatherium  in  process  of  division 92 

72.  Diffuse  nucleus  in  Chromatium  okenii  and  Beggiatoa  alba 93 

73.  B.  butschlii  in  division 95 

74.  B.  sporonema  in  spore  formation  with  vestiges  of  ancestral  sexuality  .    .  96 

75.  B.  radicosus  with  nuclear  appearances 96 

76.  B.flexilis  in  division  of  cell  and  formation  of  spores 98 

77.  Retrogression  of  original  nucleus  and  formation  of  diffuse  nucleus  in  var- 

ious bacteria 98 

78.  Life  cycle  of  Azotobacter 100 

79.  Differentiation  of  metachromatic  corpuscles  in  various  bacteria  by  means 

of  stains 102 

80.  Structure  of  bacterial  membrane  in  section 103 

81.  Capsules  (Bad.  pneumonia) 104 

82.  Distribution  of  nuclear  substance  and  various  flagella 105 

83.  Monotrichous  bacteria  (Msp.  comma) 105 

84.  Monotrichous  bacteria  (Ps.  pyocyanea) 105 

85.  Lophotrichous  bacteria  (Ps.  syncyanea) 105 

86.  Lophotrichous  bacteria  (Sp.  rubrum)   . 105 

87.  Peritrichous  bacteria  (B.  typhosus) 105 

88.  Crenothrix  polyspora 109 

89.  Spirophyttumferrugineum.Gallionellaferruginea^Leptothrixochracea...    .  no 

90.  Pasteur-Chamberland  or  Berkefeld  filtering  apparatus 120 

91.  Amceba  vespertilio 124 

92.  Paramecium  caudatum  dividing  without  mitosis 127 

93.  Stages  in  division  of  Amoeba  poly  podia 128 

94.  Multiplication  of  Coccidium  schubergi 129 


LIST    OF    ILLUSTRATIONS  XXV11 

95.  Herpetomonas  musca-domestica 134 

96.  Trypanosoma  tinea  and  Trypansoma  percce 135 

97.  Trichomonas  eberthi 136 

98.  Lamblia  intestinalis 137 

99.  Development  of  sporozoits  in  Laverania  malar  ice 138 

100.  Solutions,  diagrammatic 157 

101.  Movement  of  electric  current  and  ionization 159 

102.  Apparatus  employed  in  determination  of  H-Ion  concentration  ...'..  166 

103.  Illustrating  surface  forces 169 

104.  Illustrating  surface  pull.    .    v 170 

105.  Particle  in  Brownian  motion 172 

106.  Plasmolysis  in  cells 177 

107.  An  arrangement  of  dispersoids 181 

1 08.  Comparison  of  particles  of  different  size 182 

109.  Ultramicroscope 183 

no.  Illustrating  cell  activities 196 

in.  Amoeba  proteus 197 

112.  Influence  of  oxygen  on  microorganisms 229 

113.  Crystals  of  bacteriopurpurin 247 

114.  Carbon  cycle 259 

115.  Nitrogen  cycle 260 

116.-  Sulphur  cycle 261 

117.  Action  of  light  on  bacteria 278 

118.  Action  of  light  on  molds 279 

119.  Action  of  light  on  mold  colonies 280 

120.  Chemotaxis 286 

121.  Curve  of  disinfection 289 

122.  Influence  of  filtered  water  on  typhoid  fever  and  Asiatic  cholera 315 

123.  Section  of  sand  filter 323 

124.  Unglazed  porcelain  filters 325 

125.  126,  127.  Location  of  wells  on  farm 327 

128.  Construction  of  model  well 328 

129.  Trickling  filter,  sand  filter,  dosing  tank,  septic  tank 341 

130.  Septic  tank 342 

131.  Non-symbiotic  nitrogen-fixing  organism  (B.  pastcurianus] 402 

132.  Non-symbiotic  nitrogen-fixing  organism  (.4 zotobacter  vineland'i) 403 

133.  Ps.  radicicola 407 

134.  Section  through  root  tubercle 408 

J35)  J36,  137.  Influence  of  Ps.  radicicola 411,412,413 

138.  Section  of  cow's  udder 434 

139.  Bacterial  colonies  in  dust  from  udder 437 

140.  Bacterial  colonies  from  cow's  hair 438 

141.  Bacterial  colonies  from  dust  of  stable 439 

142.  Small-top  milk  pails 442 

143.  Ropy  cream 464 

144.  Ropy  cream  organisms 465 

145.  Chart  of  Rochester  milk  supply 469 

146.  Gassy  cheese 488 

147.  Cheese  from  lactic  starter ^ 489 

148.  Influence  of  lactic  organisms  on  casein  degradation 495 

149.  Swiss  cheese 500 

150.  Kefir  grain 509 

151.  Chart.     Effect  of  storage  on  bacterial  content  of  ice  cream 514 

152.  Chart.     Influence  of  temperature  on  sterilizing  time 537 

I53-  Chart.     Influence  of  number  of  spores  on  sterilizing  time 537 

154.  Chart.     Influence  of  speed  of  rotation  on  heat  penetration 538 

155.  Tubes  for  feces  examination 602 


XXV111  LIST    OF    ILLUSTRATIONS 

156.  Bacteria  of  slimy  wine 610 

157.  Bacteria  of  wine  diseases 611 

158.  Vinegar  bacteria 638 

159.  Vinegar  barrel 642 

160.  Rapid  process  vinegar  apparatus 645 

161.  Oidium  albicans 776 

162.  Oidium  albicans.     (Kohle  and  Wassermann.) 776 

163.  Trichophyton  tonsurans 777 

164.  165.  Actinomyces  bovis 779,  780 

166.  Gonococci .    .    .    .  ^ 785 

167.  Bact.  anthracis,  thread  formation " 803 

168.  Bact.  anthracis,  spores .    .    .    . 803 

169.  Organisms  of  anthrax  in  capillaries 804 

170.  Bact.  diphtheria 813 

171.  Wesbrook's >  types  of  Bact.  diphtheria 814 

172.  Bact.  mallei 821 

173.  Bact.  pestis 831 

174.  Bact.  tuberculosis,  branching  forms 836 

175.  Bact.  tuberculosis,  from  sputum 836 

176.  Bact.  tuberculosis,  in  culture 837 

177.  B.  tetani,  with  spores 843 

178.  B.  typhosus 848 

179.  Msp.  comma 852 

1 80.  Msp.  comma  colonies  in  gelatin 853 

181.  Kidneys  in  hog  cholera,  hemorrhagic  points 86 1 

182.  Negri  bodies 872 

183.  Amoeba  coli _ 877 

184.  Leishmania  donovani ;* 880 

185.  Structure  of  trypanosome 882 

186.  Trypanosoma  gambiense 883 

187.  Glossina  palpalis 884 

1 88.  Colonization  in  Trypanosoma  lewisi 887 

189.  Malarial  parasite  in  human  and  mosquito  cycles 891 

190.  Longitudinal  section  of  Anopheles 893 

191.  Babesia  bigemina 895 

192.  Ornithodoros  moubata 901 

193.  Spirochoeta  duttoni 902 

194.  Treponema  pallidum 903 

195.  Ps.  medicaginis 952 

196.  Pear  blight 958 

197.  Walnuts  affected  by  bacteriosis 964 

198.  Crown  gall 966 

199.  Roots  of  cabbage  plant  affected  with  " stump-root." 970 

200.  Plasmodiophora  brassica 971 

Colored  Plate 
The  Malarial  parasites 891,892 


HISTORY  OF  MICROBIOLOGY* 


Geronimo  Fracastorio,  of  Verona,  was  born  in  1484,  studied  medicine 
in  Padua,  and  published  a  work  in  Venice  in  1546,  which  contained  the 
first  statement  of  the  true  nature  of  contagion,  infection,  or  disease 
organisms,  and  of  the  modes  of  transmission  of  infectious  disease.  He 
divided  diseases  into  those  which  infect  by  immediate  contact,  through 
intermediate  agents,  and  at  a  distance  through  the  air.  Organisms 
which  cause  disease,  called  Seminaria  conlagionum,  he  supposed  to  be 
of  the  nature  of  viscous  or  glutinous  matter,  similar  to  the  colloidal 
states  of  substances  described  by  modern  physical  chemists.  These 
particles,  too  small  to  be  seen,  were  capable  of  reproduction  in  ap- 
propriate media,  and  became  pathogenic  through  the  action  of  animal 
heat.  Thus  Fracastorius,  in  the  middle  of  the  sixteenth  century,  gave 
us  an  outline  of  morbid  processes  in  terms  of  microbiology. 

Athanasius  Kircher,  in  1659,  demonstrated  the  presence  of  "minute 
living  worms  in  putrid  meat,  milk,  vinegar,  etc.;"  but  he  did  not 
describe  their  form  and  character,  and  it  is  doubtful  whether  he  ever 
saw  microorganisms. 

In  the  year  1683  Antonius  van  Leeuwenhoek,  a  Dutch  naturalist  and 
a  maker  of  lenses,  communicated  to  the  English  Royal  Society  the  re- 
sults of  observations  which  he  had  made  with  a  simple  microscope  of 
his  own  construction,  magnifying  from  100  to  150  times.  He  found  in 
water,  saliva,  dental  tartar,  etc.,  what  he  termed  "animalcula."  He 
described  what  he  saw,  and  by  his  drawings  showed  both  rod-like  and 
spiral  forms,  both  of  which,  he  said,  had  motility.  In  all  probability, 
the  two  species  he  saw  were  those  now  recognized  as  Bacillus  buccalis 
maximus  and  Spirillum  sputigenum.  Leeuwenhoek's  observations 
were  purely  objective  and  in  striking  contrast  with  the  speculative 
views  of  M.  A.  Plenciz,  a  Viennese  physician,  who  in  1762  published  a 
germ  theory  of  infectious  diseases.  Plenciz  maintained  that  there 
was  a  special  organism  by  which  each  infectious  disease  was  produced, 

•Prepared  by  F.  C.  Harrison. 


2  HISTORY   OF   MICROBIOLOGY 

that  microorganisms  were  capable  of  reproduction  outside  of  the  body, 
and  that  they  might  be  conveyed  from  place  to  place  by  the  air. 

The  important  role  that  the  compound  microscope  has  played  in 
microbiology  calls  for  something  regarding  the  invention  of  this  in- 
strument— an  invention  which  antedates  Leeuwenhoek's  discovery  by 
nearly  100  years. 

The  first  compound  microscope  was  made  by  Hans  Jansen  and  his 
son  Zaccharias,  in  1590,  at  Middelburg,  in  Holland.  The  instrument 
was  composed  of  two  lenses  mounted  in  tubes  of  iron;  a  representation 
of  it,  made  from  the  original  and  still  kept  at  Middelburg,  is  shown 
in  Fig.  i.  From  that  date  the  microscope  gradually  improved.  In 
1844  the  immersion  lens  was  introduced  by  Dolland.  In  1870  Abbe 
brought  out  the  substage  condenser,  which  still  bears  his  name.  Apo- 
chromatic  lenses  and  many  minor  improvements  were  introduced  by 
the  firm  of  Zeiss  about  1880. 


I 


FIG.  i. — Longitudinal  section  of  a  compound  microscope  made  by  Zaccharias 
Jansen  (1590).     a,  Microscope  tube;  b,  objective  tube;  c,  ocular. 

In  1786  O.  F.  Miiller  (a  Dane)  first  attempted  to  classify,  according 
to  theLinnean  system,  the  various  organisms  previously  discovered,  and 
characterized  four  or  five  genera — among  them,  the  genus  Vibrio,  in 
which,  under  the  terms  bacillus,  lineola,  and  spirillum,  we  recognize 
forms  that  correspond  with  our  "bacteria." 

From  the  middle  of  the  eighteenth  century  until  well  on  into  the 
nineteenth,  the  history  of  bacteriology  is  largely  the  story  of  a  con- 
troversy between  those  who  believed  that  minute  living  organisms,  such 
as  those  above  referred  to,  were  produced  from  inanimate  substances, 
and  that  their  formation  was  spontaneous.  Philosophers,  poets,  and 
common  people  of  the  most  enlightened  nations  accepted  this  doctrine 
down  to  the  eighteenth  century.  The  hypothesis  regarding  this  forma- 
tion was  known  as  that  of  "spontaneous  generation,"  "heterogenesis," 
and  "  abiogenesis."  The  opponents  of  this  theory  denied  the  possibility 
of  a  transition  from  a  lifeless  to  a  living  condition,  and  contended  that 
all  life  came  from  preexisting  life — a  theory  aphoristically  summed 
up  in  the  phrase  "omne  vivum  ex  vivo."  Such  was  the  doctrine  of 
Biogenesis — life  only  from  life. 


HISTORY   OF   MICROBIOLOGY  3 

In  1668,  Francisco  Redi,  an  Italian,  distinguished  alike  as  scholar, 
poet,  physician,  and  naturalist,  expressed  the  idea  that  life  in  matter  is 
always  produced  through  the  agency  of  preexisting  living  matter;  but 
the  beginnings  of  the  real  controversy  date  from  the  publication  of 
Needham's  experiments  in  1745.  The  English  divine  boiled  some  meat 
extract  in  a  flask,  made  the  flask  air-tight,  and  left  it  for  some  days. 
When  the  flask  was  opened,  he  found  in  it  what  he  termed  "infusoria." 
He  naturally  concluded  that  all  life  had  been  killed  by  boiling;  and, 
as  the  entrance  of  fresh  life  from  the  outside  was  prevented  by  the 
closing  of  the  flask,  he  considered  that  the  living  infusoria  must  have 
originated  spontaneously  from  the  inanimate  constituents  of  the  broth. 

Twenty  years  later  Abbe  Spallanzani  alleged  that  the  development 
of  the  infusoria  "in  an  infusion  maintained  at  boiling-point  for  three- 
quarters  of  an  hour  was  possible  only,  provided  air,  which  had  not  been 
previously  exposed  to  the  influence  of  fire,  had  been  admitted."  Ob- 
jections were  made  to  these  experiments  and  the  controversy  went 
merrily  on.  Gradually  experimental  evidence  accumulated — resulting 
largely  from  the  work  of  Franz  Schulze,  and  the  discovery  by  Schroeder 
and  Dusch  in  1853,  that  putrescible  fluids  will  not  decay  after  boiling,  if 
protected  from  the  bacteria  of  the  air  by  means  of  a  cotton-wool 
filter  or  plug;  and  the  epoch-making  experiments  of  Pasteur  in  1860, 
with  the  now  well-known  Pasteur  flask,  showed  conclusively  that  the 
hypothesis  of  spontaneous  generation,  or  abiogenesis,  could  not  be 
proved. 

Liebig,  the  celebrated  German  chemist,  strenuously  opposed  the 
theories  of  Pasteur;  his  authority  and  the  brilliancy  of  his  expositions 
influenced  the  scientific  world  during  the  period  1840-60.  To  Liebig, 
fermentation  was  a  purely  chemical  phenomenon  unassociated  with  any 
vital  process;  and  he  treated  Pasteur's  results  with  disdain.  "Those 
who  pretend  to  explain  the  putrefaction  of  animal  substance  by  the 
presence  of  microorganisms,"  he  wrote,  "reason  very  much  like  a  child 
who  would  explain  the  rapidity  of  the  Rhine  by  attributing  it  to  the 
violent  motions  imparted  to  it  in  the  direction  of  Bingen  by  the  numer- 
ous wheels  of  the  mills  of  Mayence."  Again  and  again  Liebig  formally 
denied  the  correctness  of  Pasteur's  assertions;  finally  Pasteur  challenged 
him  to  appear  before  the  Academic  Commission  to  which  they  would 
submit  their  respective  results.  Liebig,  however,  did  not  accept  the 
challenge;  the  victory  was  with  the  French  savant. 


4  HISTORY   OF   MICROBIOLOGY 

In  1841  Fuchs  investigated  some  blue  and  yellow  milk.  He  exam- 
ined it  with  the  microscope  and  discovered  the  presence  of  organisms. 
He  succeeded  in  cultivating  the  "blue  milk"  microbe  in  mallow  slime, 
and  re-developed  the  blue  color  in  milk  by  introducing  some  of  his 
culture.  The  organisms  obtained  were  sent  to  Ehrenberg,  who  named 
them  Bacterium  syncyaneum,  now  known  as  B.  cyano genus,  Ps.  syn- 
cyanea  and  B.  synxanthus,  a  name  which  is  still  retained  in  the 
literature. 

Since  1860  the  master  mind  of  Louis  Pasteur  has  dominated  the 
realm  of  microbiology.  His  epoch-making  discoveries  were  largely  due 
to  his  intuitive  vision,  his  skill  in  device  and  in  the  adaptation  of  means 
to  ends,  his  prodigious  industry,  and  the  enthusiasm  and  love  with  which 
he  inspired  his  associates.  Trained  as  a  chemist,  his  first  appointment 
was  to  a  professorship  of  chemistry,  and  his  earliest  research  dealt  with 
problems  in  molecular  chemistry  and  physics.  On  his  being  elected 
Dean  of  the  Faculty  of  Sciences  at  Lille,  he  commenced  to  study  fer- 
mentation. His  work  in  this  field  was  soon  followed  by  important 
results :  the  discovery  of  the  organisms  which  produce  lactic  and  butyric 
fermentation,  and  of  \  anaerobic  life,  or  life  which  flourishes  without 
free  oxygen.  He  devised  an  improved  method  of  making  vinegar,  and 
demonstrated  the  presence  of  the  acetic  organism  which  he  named 
Mycoderma  aceti.  Later  he  studied  the  diseases  •  of  wine,  and  dis- 
covered that  bitterness  or  greasiness  was  due  to  a  special  ferment,  and 
suggested  the  heating  of  wines  in  closed  bottles  to  a  temperature  of 
60°,  in  order  to  kill  the  injurious  microorganisms.  This  process,  since 
called  pasteurization,  is  now  largely  used,  and  makes  it  possible  for 
manufacturers  and  merchants  to  keep  and  export  wine  without  losing 
its  flavor  or  bouquet.  It  is  interesting  in  this  connection  to  note  that 
a  French  confectioner  named  Appert  published,  in  1811,  his  method  of 
preserving  fruits,  vegetables,  and  liquors  by  heating  and  sealing,  and 
hence  may  be  looked  upon  as  the  founder  of  the  packing  and  canning 
industry. 

In  1864-65  the  silk  districts  of  that  region  of  France,  known  as  the 
Midi,  suffered  such  serious  losses  that  the  yield  of  cocoons  fell  from 
twenty-six  million  kilograms  to  four  million,  which  entailed  a  loss  of 
twenty  million  dollars  and  caused  widespread  distress  and  poverty. 
An  epidemic  had  broken  out  among  the  silk-worms — the  dread 
disease  known  as  Pebrine.  Pasteur  was  induced  to  make  an  in- 


HISTORY   OF   MICROBIOLOGY        .  5 

vestigation  as  to  the  best  means  of  combating  the  epidemic;  and,  after 
several  years  of  study,  he  found  the  organism  causing  the  disease, 
suggested  remedies,  and  brought  back  wealth  to  the  ruined  com- 
munities, but  at  the  cost  to  himself  of  impaired  health  and  partial 
paralysis. 

Pasteur's  results  were  very  suggestive;  and  one  outcome  of  his  work 
was  that  between  1870  and  1880  several  important  discoveries  were 
made  by  other  investigators.  Prior  to  the  dates  mentioned,  the 
mortality  from  blood  poisoning,  gangrene,  and  other  infections  follow- 
ing operations  was  extremely  high.  Surgeons  regarded  such  a  result 
as  inevitable,  and  many  agreed  with  the  saying  of  Velpeau,  that  "the 
prick  of  a  pin  is  the  open  door  to  death;"  but,  in  1860,  Joseph  Lister, 
an  Edinburgh  surgeon,  began  to  study  the  possible  role  of  microbes  in 
the  infection  of  wounds.  By  sterilizing  his  instruments,  sponges,  liga- 
tures, etc.,  and  using  antiseptics,  he  was  able  to  obtain  such  a  high 
percentage  of  recoveries  that  in  two  years  he  saved  thirty-four  patients 
out  of  forty — a  percentage  unheard  of  up  to  that  time.  Hence  the 
origin  of  the  antiseptic  and  aseptic  methods  of  surgery  is  traceable 
to  Lister's  efforts.  Lister's  methods,  suggested  by  the  ideas  of  Pas- 
teur, have  rendered  possible  the  marvelous  surgery  of  the  present  day, 
banished  hospital  gangrene,  and  robbed  confinement  of  its  terrors. 

To  Lister  must  also  be  given  the  honor  of  devising  the  first  practical 
way  of  obtaining  a  pure  culture  of  bacteria  by  means  of  high  dilutions. 
By  using  this  method,  Lister  obtained  some  idea  of  the  different  fer- 
mentations of  milk,  such  as  souring,  curdling,  etc.  He  also  confirmed 
the  conclusion  of  Robert  Hall  (1874),  that  milk  could  be  obtained 
from  the  animal  in  a  sterile  condition,  thus  proving  that  the  souring 
of  milk  was  caused  by  organisms  from  some  external  source. 

In  1872,  F.  Cohn's  System  of  Classification,  based  on  morphological 
characters,  appeared.  He  distinguished  six  genera — micrococcus,  bac- 
terium, bacillus,  vibrio,  spirillum,  and  spirochaete;  four  years  later  this 
investigator  made  the  important  discovery  of  endospores  (spores  formed 
within  cells),  and  noticed  that  organisms  in  this  state  were  more  re- 
sistant to  heat  than  the  rods  from  which  they  were  derived.  This  fact 
was  observed  in  the  well-known  "hay  bacillus." 

In  1871,  Weigert  succeeded  in  staining  bacteria  with  picro-carmine ; 
but  it  was  not  until  1876  that  he  used  the  aniline  colors,  or  dyes,  for  this 
purpose,  and  thus  opened  up  a  new  field  which  was  exploited  with  such 


6  HISTORY   OF  MICROBIOLOGY 

beautiful  results  by  Ehrlich,  Koch,  Gram,  and  others.  The  staining 
of  microorganisms  rendered  it  possible  to  obtain  pictures  of  them  by 
photographic  methods;  the  art  of  photomicrography  developed  thus 
rapidly. 

In  1879,  Miquel  discovered  bacteria  which  grew  or  developed  at  tem- 
peratures between  65°*  and  75°.  He  isolated  them  first  from  the  waters 
of  the  Seine,  and  subsequently  from  dust,  manure,  and  other  substances. 
Later  researches  have  shown  that  these  thermophilic  organisms  play  im- 
portant roles  in  various  fermentations. 

The  ninth  decade  of  the  last  century  was  prolific  in  important  bac- 
teriological events.  Discovery  followed  discovery  in  rapid  succession. 
In  1880,  Laveran,  a  French  military  surgeon,  discovered  the  protozoon  of 
malaria;  in  1881  Robert  Koch  introduced  the  poured  gelatin  and  agar 
plate,  which  made  it  possible  to  obtain  pure  cultures  without  difficulty. 
Investigators  were  quick  to  take  advantage  of  this  method  and  notable 
results  followed.  Eberth  and  GafTky  discovered  the  bacillus  of  typhoid 
fever,  and  succeeded  in  growing  it  in  culture  media.  In  1882,  Loeffier 
and  Schiitz  discovered  the  bacterium  which  causes  glanders ;  and  in  the 
following  year  Koch  isolated  the  vibrio  of  Asiatic  cholera  from  the  in- 
testines of  cholera  patients.  In  1883  Klebs  described  the  diphtheria 
bacterium;  and,  in  1884,  Loeffler  grew  the  organism  in  pure  culture. 

In  1884,  Koch  published  his  results  on  the  etiology  of  tuberculosis, 
in  a  paper  which  will  remain  as  a  classical  masterpiece  of  bacteriological 
research,  owing  to  the  difficulty  of  the  task  and  the  thoroughness  of  the 
work.  Not  only  did  Koch  show  the  tubercle  bacterium  by  appropriate 
staining  methods,  but  he  succeeded  in  obtaining  pure  cultures  of  it  and 
in  producing  tuberculosis  by  inoculation  with  his  isolated  cultures. 

In  1885,  Nicolaier  observed  the  tetanus  bacillus  in  pus  produced  by 
inoculating  mice  and  rabbits  with  soil;  later,  in  1889,  Kitasato  isolated 
this  organism,  and  showed  that  the  cause  of  the  failure  in  earlier 
attempts  to  isolate  it  were  due  to  the  fact  that  it  could  grow  only  in  the 
absence  of  free  oxygen.  The  specific  infecting  agents  in  pneumonia 
were  discovered  by  Friedlander  and  Fraenkel  about  this  time,  as  were 
also  several  organisms  associated  with  inflammation  and  suppuration, 
such  as  the  Streptococcus  pyogenes  and  the  Staphylococcus  pyogenes, 
discovered  by  Rosenbach,  and  the  green  pus  germ  (Pseudomonas 
pyocyanea)  by  Gessard. 

*A11  temperatures  are  stated  in  Centigrade  scale,  unless  otherwise  indicated. 


HISTORY    OF   MICROBIOLOGY  7 

While  these  discoveries  were  taking  place,  largely  in  Germany,  Pas- 
teur had  been  engrossed  with  his  prophylactic  studies.  In  1880,  he  dis- 
covered a  method  of  vaccination  against  fowl  cholera;  and  in  1881  he 
published  his  method  of  vaccination  against  anthrax.  On  a  farm  at 
Pouilly  le  Fort,  sixty  sheep  were  placed  at  Pasteur's  disposal;  ten  of 
these  received  no  treatment,  and  twenty-five  were  vaccinated.  Some 
days  afterward  the  latter  were  inoculated  with  virulent  anthrax,  and  also 
twenty-five  which  had  received  no  vaccine.  The  twenty-five  non- 
vaccinated  sheep  died,  and  the  twenty-five  vaccinated  ones  remained 
healthy  and  in  the  same  state  as  the  ten  control  animals.  This  con- 
vincing experiment  was  followed  by  others;  and,  in  the  twenty-five 
years  immediately  following  the  introduction  of  the  method,  more 
than  ten  million  animals  were  vaccinated  in  France  alone,  with  ex- 
cellent results.  In  1885,  as  the  result  of  much  animal  experimentation, 
Pasteur  related  to  the  Academy  of  Sciences  his  discovery  of  a  method 
of  vaccination  against  rabies,  or  hydrophobia;  and  six  months  after 
the  successful  treatment  of  the  first  case,  350  persons  bitten  by  rabid 
dogs  were  vaccinated.  An  institute  for  the  preparation  of  vaccines 
was  built  by  public  subscription  and  named  the  Pasteur  Institute;  and 
since  that  date  more  than  thirty  similar  establishments  have  been 
founded  in  different  parts  of  the  world. 

This  eighth  decade,  so  pregnant  with  discoveries  of  the  utmost  im- 
portance to  medicine  and  surgery,  was  also  notable  for  its  discoveries  in 
agricultural  bacteriology.  The  honor  of  having  been  the  first  to  work 
out  the  causal  relation  between  a  specific  microbe  and  a  plant  disease 
belongs  to  Burrill,  who  discovered  the  organism  of  Fire  or  Pear  Blight; 
and  in  1883  to  1888  Wakker  discovered  the  bacillus  which  produces  the 
" yellows"  of  the  hyacinth,  a  disease  of  considerable  economic  im- 
portance in  Holland.  To  Beyerinck,  Hellriegel,  and  Wilfarth  we  owe 
our  earlier  knowledge  of  the  development  and  morphology  of  the 
nitrogen-fixing  organism  which  produces  the  nodules  or  tubercles  on 
the  roots  of  legumes.  In  1888  Winogradsky  isolated  from  soils  nitrify- 
ing microbes  which  grew  in  a  medium  devoid  of  all  traces  of  organic 
matter.  During  this  period,  Hansen's  investigations  along  the  line  of 
the  fermentation  industry  were  most  important.  He  devised  methods 
for  securing  pure  cultures  of  yeasts  starting  from  a  single  cell,  showed 
that  yeasts  produced  diseases  in  beer,  and  established  the  method  of 


8  HISTORY    OF   MICROBIOLOGY 

identifying  yeasts  by  observing  their  microscopic  appearance,  the  for- 
mation of  ascospores,  and  the  production  of  films. 

The  tenth  decade  of  the  nineteenth  century  was  almost  as  prolific  in 
discovery  as  the  ninth.  In  1890  Behring  discovered  the  antitoxin  for 
diphtheria,  as  a  result  of  the  pioneer  work  on  toxins  by  Roux  and 
Yersin.  Five  years  later,  this  serum  came  into  general  use  as  a  cura- 
tive agent;  and  the  efficiency  of  the  treatment  is  shown  by  a  comparison 
of  the  death  rate  from  diphtheria  before  and  after  the  introduction 
of  the  antitoxin.  The  average  annual  death  rate  from  diphtheria  in 
eight  large  cities,  during  the  period  1885-94,  was  9.74  per  10,000  of 
the  population  before  the  use  of  antitoxin;  and  during  the  antitoxin 
period  of  1895-1904  it  was  4.29. 

The  subsequent  researches  on  the  constitution  of  toxins  and  anti- 
toxins by  Ehrlich,  MetchnikofI,  Madsen,  and  others  have  been  pro- 
ductive of  a  better  understanding  of  the  problems  of  immunity. 

In  1892  Pfeiffer  discovered  the  organism  of  influenza  or  grippe;  and 
in  1894  Yersin  and  Kitasato  independently  discovered  the  bacterium  of 
bubonic  plague. 

The  now  well-known  serum  diagnosis  of  typhoid  fever,  whereby 
living  and  motile  typhoid  bacilli  are  clumped  and  lose  their  motility 
when  placed  in  the  diluted  serum  of  a  patient  suffering  from  the 
fever,  was  due  to  the  work  of  Gruber  and  Durham,  and  the  exploitation 
of  the  method  by  Widal  dates  from  1896. 

In  1898,  Shiga  discovered  the  bacterium  of  dysentery,  and  the  pos- 
sible cause  of  pleuro-pneumonia  in  cattle  was  found  by  Nocard.  This 
latter  organism  was  so  minute  as  to  be  at  the  extreme  limit  of  micro- 
scopic definition,  and  suggested  that  other  well-known  diseases,  such  as 
foot-and-mouth  disease,  are  probably  caused  by  ultra-microscopic 
organisms. 

This  year,  Ronald  Ross  worked  out  the  relation  between  man,  the 
mosquito,  and  the  malarial  parasite — a  discovery  which  at  once  sug- 
gested the  best  means  of  controlling  the  disease. 

In  1905,  Schaudinn  definitely  established  the  causal  agent  of  syphi- 
lis, a  spirochaete-shaped  organism,  which  he  named  Treponema  pallidum, 
and  which  had  escaped  earlier  discovery  on  account  of  its  being  refractory 
to  the  ordinary  staining  methods. 

In  the  last  decade,  our  knowledge  of  certain  communicable  diseases 
has  been  extended  considerably.  Preventive  and  prophylactic  measures 


HISTORY    OF    MICROBIOLOGY  Q 

have  been  studied  extensively  and  carried  out  on  a  scale  never  before 
contemplated,  and  probably  made  possible  only  by  war  conditions.  A 
few  of  these  may  be  mentioned  as  examples  of  the  progress  made: — 
the  Dakin-Carrel  treatment  of  septic  wounds,  the  immunization  of 
troops  against  typhoid,  tetanus  and  pneumonia;  the  increasing  use, 
improvement  in  manufacture  and  efficacy  of  protective  and  curative 
sera  and  vaccines;  the  importance  of  the  carrier  in  many  infections, 
and  the  means  whereby  he  is  dealt  with,  as  instanced  in  the  case  of 
infection  with  the  meningococcus;  the  discovery  of  filtrable  viruses  as, 
to  quote  the  most  recent  (1919),  the  inciting  agent  of  mumps. 

No  one  can  deny  that  the  progress  of  microbiology  in  the  last  fifty 
years  has  been  wonderful,  and  in  the  last  few  years  extraordinary,  but 
much  still  remains  unknown  and  new  problems  appear  from  time  to 
time.  The  etiology  of  certain  diseases  yet  remain  undiscovered.  The 
cause  of  the  disease  known  as  influenza  which  carried  off  so  many  in 
the  fall  of  1918  remains  as  yet  unknown  although  some  reports  of 
alleged  discoveries  have  been  made.  Trench  fever  is  another  example 
of  a  problem  suddenly  appearing  and  necessitating  instant  solution. 

"In  the  last  few  years  a  group  of  pleomorphic  organisms  have 
been  discovered,  which  are  associated  with  typhus,  Rocky 
Mountain  fever  and  trench  fever.  These  organisms  are  carried  by 
insects  but  have  not  yet  been  cultivated." 

So  also  with  other  fields  of  research.  Great  progress  has  been  made 
in  water  and  food  microbiology;  more  attention  is  being  paid  to  parasi- 
tology;  soil  organisms  and  especially  soil  protozoa  are  receiving  more 
study  and  our  technique  has  advanced  with  great  strides. 

In  short  the  work  of  the  microbiologist  has  become  of  increasing 
interest  and  importance  in  all  lines  of  work. 

The  record  of  past  achievement  is  an  inspiration;  and  the  knowledge 
that  each  discovery  is  the  result  of  persistent  and  concentrated  effort, 
may  give  us  of  the  present  day  firmer  faith  and  greater  strength  for 
work  in  the  broad  and  inviting  field  outlined  in  this  text  book. 


PART  I 

THE  MORPHOLOGY  AND  CULTURE  OF  MICRO- 
ORGANISMS 


GENERAL* 

Microbiology  is  concerned  with  organisms  which  range  between 
well  defined  plant  life  on  the  one  hand,  and  well  defined  animal  life 
on  the  other.  These  living  forms  are  in  the  main  unicellular  in 
structure.  A  gradation  exists  from  the  plant  world  into  this  mi- 
crobe-world and  also  from  the  animal  world.  No  sharp  lines  can 
be  established  because  Nature  seems  to  blend  from  one  type  into 
another  leaving  no  particularly  characteristic  barrier,  although  man, 
for  his  own  convenience,  strives  to  construct  Nature  with  very 
definite  lines  of  demarcation.  Haeckel  was  so  impressed  with  the 
organisms  which  lie  between  the  animal  and  plant  world  that  he 
found  it  undesirable  to  attempt  to  classify  them  in  the  one  or  the 
other  kingdom.  Accordingly,  he  believed  it  of  sufficient  importance 
to  give  a  specific  name,  Protista,  to  the  microorganisms  included  in 
this  specific  kingdom.  This  relationship  is  clearly  set  forth  by  an 
illustration  furnished  by  Minchinf  (Fig.  2). 

Morphology  has  been  paramount  in  classification  in  the  past,  yet, 
at  first,  bacteria  were  called  animals  and  later  plants.  With  the  ad- 
vancement and  importance  of  physiology,  it  becomes  necessary  to 

*  Editor. 

t  Minchin,  B.  A.*  An  Introduction  to  the  Study  of  the  Protozoa. 

II 


12  MORPHOLOGY   AND   CULTURE    OF   MICROORGANISMS 

consider  physical,  chemical,  nutritive  or  digestive  and  general  physiolo- 
gical processes  along  with  morphological  characters.  When  these  are 
considered  there  is  a  marked  resemblance  of  microorganisms,  even 
molds  and  yeasts,  to  animal  life.  Assignment  to  either  animal  or  plant 
life  is  precarious  and  unnecessary,  for  in  making  such  an  attempt  the 
scientist  really  does  nothing  more  than  prescribe  for  Nature  restrictions 
rather  than  follow  Nature  as  she  exists. 


FIG.  2. — Graphic  representation  of  the  relation  of  the  animal  and  vegetable 
kingdoms  to  the  kingdom  of  Protista  (Protistenreicti) .  The  Protozoa  are  represented 
by  the  portion  of  the  triangle  representing  the  animal  kingdom  which  lies  within 
the  circle  representing  the  Protista.  (After  Minchin.) 


From  the  organization  of  microbiology  by  Pasteur,  the  technic  of 
the  subject  together  with,  in- large  part  as  well,  its  economic  bearing 
seems  to  be  the  applied  determining  factor  in  bounding  the  field.  The 
subject  of  microbiology  is  following  at  present  the  course  of  all  scien- 
tific branches — it  is  undergoing  division  for  purposes  of  intensification 
demanded  by  practice  and  by  the  limitations  of  man's  capacity. 


OUTLINE    OF   PLANT    GROUPS  13 

OUTLINE  OF  PLANT  GROUPS* 

The  following  is  a  diagram  of  plant  groups,  showing  one  scheme  of 
placing  the  bacteria,  yeasts,  and  molds  in  relation  to  other  groups. 
Only  a  few  of  the  sub-groups  can  be  shown  in  such  a  scheme. 


Plants 


Schizophyta 
(fission-plants) 


I  Schizomycetes  (fission-fungi),  bacteria. 

I  Schizophyceae  (fission-algae),  blue-green  algae. 

Chlorophyceae — green  algae. 
Algae        Phaeophyceae — brown  algae. 

.  Rhodophyceae — red  algae. 
Characeae. 

Myxomycetes. 
Actinomycetes 


Thallophyta 


Fungi 


Phycomycetes 


Chytridineae. 
Zygomycetes 

Oomycetes 


(Mucors). 
Saprolegniaceae. 

(water  fungi). 
Peronosporaceae. 

(downy  mildews). 


Ascomycetes 


Imperfect  Fungi, 
Conidia  only 

Basidiomycetes 


Hemiasci  (Monascus). 

Protoascineae   (Saccharo- 

myces,  Yeasts). 
Protodiscineae. 
Euasci      Discomycetes. 

Plectascineae      (Aspergil- 
lus,  certain  Penicillia) 
Pyrenomycetinese. 
f  Penicillium,  Fusarium,  Alternaria.f 
I  Oidium,  Cladosporium,  and  others. 
Rusts. 
Smuts. 
Mushrooms. 


Bryophyta  (mosses  and  liverworts). 
Pteridophyta  (ferns,  etc.) 
Spermatophyta  (seed  plants). 


t  Ascomycetous  species  occur  among  these  genera  but  such  species  are  rarely  met  in  bacteriol- 
ogical work;  many  of  the  common  species  of  Aspergillus  lack  the  ascigerous  form,  hence  are 
classified  by  their  conidial  forms  only. 


OUTLINE  OF  PROTOZOAL  GROUPS f 

"AN  OUTLINE  CLASSIFICATION  OF  THE  PROTOZOA,"  embracing  only  parasitic 
and  more  especially  the  forms  pathogenic  for  man  and  domestic  animals.  For 
discussion  of  classification  see  p.  133. 


Protozoa 


Rhizopoda 


Enlamaba  buccalis 

Entamasba  coll 
Entamceba       Entamceba  histolytica 

Entamceba  mehagridis 
Plasmodiophora  { Plasmodiophora  brassies. 


Charles  Thorn. 
J.  L.  Todd. 


MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 


Protozoa 


Flagellata 


Sporozoa 


Leishmania 


Crithidia 


Trypanosoma 


Leishmania  donovani 
Leishmania  tropica 
Leishmania  infantum 
Trypanosoma  gambiense 
Trypanosoma  rhodesiense 
Trypanosoma  cruzi 
Trypanosoma  brucei 
Trypanosoma  equinum 
Trypanosoma  evansi 
Trypanosoma  lewisi 
Trypanosoma  equiperdum 


Trypanoplasma 

Cercomonas 

Trichomonas 

Monas 

Plagiomonas 

Lamblia  \Lambha  intestinalis 

Gregarina 


f  Trichomonas  intestinalis 
\  Trichomonas  vaginalis 


Coccidium 


Haemosporidia 


Infusoria 


Parasites    of    uncertain 
position 


|  Eimeria  cuniculi  (Coccidium  stiedce) 
\  Eimeria  avium 

Plasmodium  vivax 
Plasmodium    Plasmodium  malaria 

Plasmodium  falciparum 
Proteosoma 
Haemoproteus 
Haemogregarina 
Hepatozoon 

Babesia  bovis  (bigemina} 
Babesia  canis 
Babesia    {  Babesia    parva 
Bartonella 
Anaplasma 

Sarcosporidia  { Sarcocystis  { Sarcocystis  miescheriana 
Haplosporidia  { Rhinosporidium  { Rhinos poridium  kinealyi 
Myxosporidia  { Myxobolus  { Myxobolus  pfeifferi 
Microsporidia  { Nosema  { Nosema  bombycis 
{  Eala,ntidi\im{Balantidium  coli 
Toxoplasma 
Histoplasma 
Chlamydozoa 
Rickettsia 
Ultramicroscopic  viruses 

j  Spirochata  recurrentis 
j  Spirochata  vincenti 
[  Spiroch(Bta  gallinarum, 
f  Treponema  pallidum 
\  Treponema  pertenuq 


Spirochaeta 


.  Treponema 


CHAPTER  I* 

ELEMENTS  OF  MICROBIAL  CYTOLOGY 
CELLS  AND  ENERGIDS 

The  microorganisms  are  confined  to  cells,  such  as  algae,  molds, 
bacteria,  yeasts,  and  protozoa,  or  cytoplasmic  masses  with  a  nucleus 
associated  with  each  (Fig.  3).  Some  are,  however,  made  up  of  rows 
of  cells,  such  as  threads  of  Cladothrix,  occasionally  capable  of  branching 
out, like  the  mycelium  of  a  mold  (Fig.  4,  A).  There  are  also  some  cells 
which  have  a  special  structure.  In  each  cell  are  enclosed  several 
nuclei.  If  certain  amoabee  are  examined,  for  example,  Pelomyxa  pa- 
lustris  (Fig.  4,  B),  inside  of  what  appears  to  be  a  cell  there  are  found 
many  nuclei.  Such  cells  have  not  the  anatomical  value  of  true  cells, 
but  seem  to  represent  as  many  cells  as  there  are  nuclei.  Each  of 
these  nuclei  with  the  cytoplasm  which  surrounds  it,  equivalent  to  a 
cell,  may  be  called  specifically  an  energid.  Some  algae  and  fungi  are 
made  up  of  threads  of  cells  enclosing  several  nuclei;  each  cell  in- 
cluded in  a  thread  consequently  represents  a  group  of  organized  ele- 
ments, the  union  of  several  energids  in  the  same  anatomical  unit  (Fig. 
4,  A). 

STRUCTURE  OF  THE  CELL 

A  typical  cell  is  constituted  of  three  essential  elements :  the  nucleus ; 
the  cytoplasm;  and  the  cell-membrane. 

The  general  characteristics  of  these  three  elements,  and,  follow- 
ing this,  the  study  of  cell  reproduction,  may  now  be  systematically 
presented. 

THE  NUCLEAR  STRUCTURE. — General  Structure  of  the  Nucleus. — The 
nucleus  frequently  takes  in  microorganisms  the  typical  form  which  it 
assumes  in  the  higher  organisms,  namely,  that  of  a  spherical  vesicle 
limited  by  a  membrane,  enclosing  a  hyaline  substance  called  the 
nuclear-fluid,  or  nucleoplasm  (Fig.  22,  A,  a,  B,  a).  In  this  nuclear 

*By  A.  Guilliermond. 

15 


1 6  MORPHOLOGY   AND    CULTURE    OF   MICROORGANISMS 

fluid  are  found:  the  nucleolus,  a  spherical  corpuscle  made  upoipyrinin 
to  which  the  chromatin,  a  characteristic  substance  of  the  nucleus,  fre- 
quently attaches  itself;  the  chromatic  network,  the  thread  of  which  is 
made  up  of  linin,  a  very  slightly  chromophilic  substance,  enclosing 
some  grains,  the  grains  of  chromatin,  which  possess  a  special  affinity 
for  basic  stains.  The  chromatin  or  nuclein  is  the  most  important 
substance  of  the  nucleus. 

Centriole. — In  intimate  contact  with  the  exterior  of  the  nucleus  and 
sometimes  inside  is  usually  found  a  small  body  called  the  centrosome, 
or,  if  the  dense  chromatin  alone  is  considered,  the  centriole  (Fig.  21, 
B,  a).  It  is  a  small  chromophilic  grain  which  is  often  surrounded  by  a 
clear  zone  of  protoplasm  called  archo plasm. 


*•*•:* 

** . 


FIG.  3.  FIG.  4. 

FIG.  3. — Cells  of  Saccharomyces  cerevisia. 

FIG.  4. — Cells  made  up  of  several  energids.  A,  A  portion  of  the  mycelium  of  a 
mold,  Aspergillus  ochraceus.  (After  Dangeard.}  B,  Cell  of  an  amoeba,  Pelomyxa 
palustris.  (After  Doflein). 

Value  of  the  Nucleus. — The  nucleus  is  an  organ  indispensable  to 
cellular  life.  It  directs  for  the  most  part  the  physiological  functions 
of  the  cell.  It  plays  an  active  part  in  nutrition  as  is  indicated  by  the 
fact  that  the  greater  part  of  the  products  of  nutrition  or  of  reserve 
spreads  itself  around  the  nuclear  membrane.  Finally,  it  assumes  an 
important  role  in  cellular  division  and  in  sexual  phenomena. 

The  experiments  of  Balbiani  which  have  been  repeated  by  other 
authors  show  that  the  cell  cannot  function  without  its  nucleus.  By 
cutting  an  infusorial  cell  in  two  portions,  one  of  which  contains  the 
nucleus  and  the  other  only  its  cytoplasm,  Balbiani  found  that  the 
nucleated  part  was  able  to  resist  the  wound  which  it  had  received 
and  regenerate  the  cytoplasm  which  was  lacking;  whereas  the  enucleated 
portion  soon  perished. 


ELEMENTS    OF   MICEOBIAL  CYTOLOGY 


FIG.  5—  Dif- 
fuse nuclei  of 
bacteria.  A,  B. 
mycoides.  (After 
Guilliermond.)  J5, 
Thiothrix  ten- 
uis.  (After 
Sivellengrebel.} 


It  does  not  seem  probable,  therefore,  that  cells  can  exist  without 
their  nuclei.     Nevertheless,  to  the  present  time  it  has  not  been  possible 
to  find  conclusive  proof  of  the  presence  of  a  true  nucleus  in  bacteria. 
The  presence  in  their  cells,  however,  of  a  great  num- 
ber of  small  chromatin  grains  like  the  chromatin  ma- 
terial of  nuclei,  and  their  evolution  during  the  forma- 
tion of  spores,  force  the  observer  to  admit  that  these 
represent  grains  of  nuclear  substance,  and  that  bac- 
teria have  a  kind  of  diffuse  nucleus,  which  is  scattered 
in  the  form  of  small  grains  (Fig.  5)  in  the  cytoplasm 
of  the  cell. 

Forms  oj  Nuclei  in  Microorganisms. — The  nucleus 
of  primitive  microorganisms  is  far  simpler  than  in 
the  higher  forms,  where  it  becomes  fairly  complex. 
Consequently  in  the  Cyanophycece  or  blue-green  algae, 
the  lowest  of  all  algae,  the  nucleus  is  in  a  very  primitive  state.  It  is 
large,  not  separated  from  the  cytoplasm  by  a  membrane,  and  is  made 
up  simply  of  a  nuclear  fluid  and  a  chromatic  network.  The  cyto- 
plasm is  confined  to  a  thin  cortical  layer 
and  the  nucleus  nearly  fills  the  cell  (Fig.  6). 
In  other  microorganisms  the  nucleus  is 
much  more  complex.  Yet  frequently  this 
nucleus  is  found  in  a  primitive  state  quite 
different  from  typical  nuclei  of  higher 
organisms.  In  some  amoebae,  the  nucleus 
is  formed  simply  of  a  poorly  defined  mem- 
brane filled  with  nuclear  fluid,  and  a  large 
body  of  chromatin  resembling  a  nucleolus 
called  the  karyosome  or  centriole-nucleolus 
(Fig.  22),  because  it  acts  both  as  a  cen- 
triole  and  as  a  nucleolus.  In  the  center  of 
the  karyosome  is  frequently  seen  a  more 
intensely  chromophilic  corpuscle  corre- 
sponding to  the  centriole  (Fig.  21,  B,  a). 

Many  protozoa  and  some  algae  have  a 

centriole-nucleolus,   but  it  is  wholly  enclosed   in   the   nuclear  fluid. 

The  chromatin  appears  as  little  grains  or  as  a  network  (Fig.  21,  A,  a). 

In  the  higher  microorganisms  (protozoa  and  fungi)   the  nucleus 


FIG.  6. — Nuclei  of  Cyano- 
phycea.  A,  Thread  of  Rvou- 
laria  bullata  with  nuclei  in 
process  of  division.  B-D, 
Fragments  of  threads  of  Calo- 
thrix  pulvinata  showing  nuclear 
division. 


1 8  MORPHOLOGY  AND   CULTURE   OF  MICROORGANISMS 

begins  to  take  the  form  of  typical  nuclei.  The  centriole  detaches 
itself  from  the  karyosome  which  becomes  a  true  nucleolus,  and  may 
remain  either  wholly  intranuclear  (Fig.  20,  A,  a,  22,  A,  a),  or  become 
entirely  extranuclear  (Fig.  20,  B,  a,  22,  B,  a). 

Theory  of  Binuclearity  of  Cells  and  Chromidia. — In  the  infusoria,  the 
nuclear  structure  divides  into  two  nuclei  (Fig.  8);  a  large  one,  the 
maeronucleus  or  vegetative  nucleus,  which  functions  during  the  vegetative 
life  of  the  cell,  and  a  small  one  lodged  in  a  hollow  of  the  maeronucleus, 
the  reproductive  nucleus  or  micronucleus.  At  fertilization,  the  macro- 
nucleus  is  disorganized  and  its  place  taken  by  the  micronucleus  which 
reproduces  by  division  both  a  micronucleus  and  a  maeronucleus. 
Certain  flagellates  have  likewise  two  nuclei,  a  large  vegetative  and  re- 
productive nucleus,  and  a  small  micro- 
or  kinetonucleus  which  controls  the  for- 
mation of  the  flagellum. 

Starting  from  these  facts,  a  few  in- 
vestigators  have   tried   to   demonstrate 

T-,.  ™       . ,.     .  that  all  cells  have  two  nuclei.     Recent 

rig.    7. — Chromidia  in  pro- 
tozoa.   A,  The  cycle  of  the  mi-     evidence  reveals   that   there  are  in  the 

wir^lS^r  £"££  cytoplasm  of  most  protozoa  sma11  chr°- 

maba  histolytica.  (After  Hart-  mophilic  granules,  like  the  chromatin 
chromidia"'  Nucleus>  chr'  material,  which  are  supposed  to  emigrate 

from  the  nucleus  during  certain  phases 

of  development,  and  which  are  likened  to  the  nuclear  substance 
(Fig.  7).  These  granules  are  called  chromidia,  and  all  the  granules 
scattered  in  the  cytoplasm  are  designated  as  the  chromidial  structure 
or  chromidium.  Chromidia  have  been  found  in  the  cells  of  higher 
organisms.  There  is  a  theory  that  this  chromidial  system  repre- 
sents a  second  nucleus,  the  vegetative  nucleus,  scattered  in  the  cyto- 
plasm, and  that  the  entire  cell  is  provided  with  two  nuclei,  one  of 
which  has  passed  unseen  up  to  this  time  because  of  its  diffuse  form. 
This  theory  is  much  doubted  to-day,  and  it  seems  probable  that  the 
chromidium  is  simply  a  reserve  material  for  the  cell,  or  corresponds 
to  formations  which  will  be  described  later  as  mitochondria. 

CYTOPLASM. — Appearance  and  Properties  of  Cytoplasm. — Cytoplasm 
may  be  denned  for  our  purposes  as  a  semi-fluid  substance,  granular  in 
appearance,  and  reacting  with  an  acid  stain.  It  has  three  essential 
physiological  properties,  nutrition,  motility,  and  sensibility.  Cyto- 


ELEMENTS    OF   MICROBIAL   CYTOLOGY  1 9 

plasm  appears  to  be  composed  largely  of  protein  substances  and  of 
diverse  lipoid  substances  in  a  state  of  colloidal  solution.  It  varies 
widely  according  to  circumstances,  consequently  it  may  be  useless  to 
search  for  any  definite  structure.  In  many  microorganisms,  as  for 
example  the  protozoa,  there  is  on  the  periphery  of  the  cell  a  hyalin  zone 
which  is  called  the  ectoplasm  to  distinguish  it  from  the  rest  of  the 
cytoplasm,  the  endoplasm  (Fig.  17). 

Chondriosomes. — Recent  research  has  demonstrated  special  func- 
tioning bodies  in  the  cytoplasm,  the  mitochondria,  which  seem  to  be 
the  constructive  elements  of  cytoplasm.  They  are  a  part  of  its  struc- 
ture, and  are  supposed  to  play  an  important  physiological  role  in  the 
cell.  These  structures,  visible  in  the  living  organism,  but  stained 


V 


FIG.  8. — Glaucoma  piriformis,  FIG.  9. — Division  of  micronu- 

infusorian   with   (N)  fmacronu-  cleus  and  of  the  chondriosomes 

cleus,     (n)     micronucleus,  (ch)  in  Carchesium  polypinum,  infu- 

mitochondria,     (vp)     pulsating  sorian.     (After  Faur6-Frtmiet.} 
vacuole.         (After     Faurt-Fri- 
miet.} 

only  by  a  special  process,  are  sometimes  in  the  form  of  small  isolated 
granules  (granular  mitochondria,  Fig.  8,  B),  or  of  small  threads  (thread- 
mitochondria)  or  sometimes  of  rods  much  like  certain  bacilli  (rod- 
mitochondria,  Fig.  8,  4).  These  forms  frequently  change  from  one  to 
the  other.  The  granular  mitochondrium  is  able  to  elongate  itself  into 
a  rod  which  is  itself  capable  of  dividing  up  into  thread-mitochondria. 
All  the  mitochondria  of  one  cell  are  called  the  chondrium.  These 
structures  seem  to  be  made  up  of  lipoidal  substance  and  phosphates  of 
albumin. 

The  mitochondria  cannot  generate  themselves  directly  from  the 
cytoplasm,  but  are  formed  always  from  preexisting  mitochondria  by 
division.  They  apparently  transmit  themselves,  after  having  divided, 
from  the  egg  to  the  adult  individual,  and  from  the  adult  individual 
to  the  egg  (Fig.  9). 


20  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

Physiologically,  mitochondria  are'  organs  of  elaboration.  In 
them,  through  some  unknown  physico-chemical  phenomena,  most  of 
the  products  of  cell  activity  may  be  formed.  The  product,  whatever 
may  be  its  specific  nature,  has  its  origin  in  a  granular  mitochondrium 
or  in  a  rod-mitochondrium.  Each  product  is  surrounded  by  a 
mitochondrial  exterior  surface  inside  of  which  it  develops  slowly;  the 
exterior  surface  remains  until  the  product  has  reached  its  state  of 
maturity. 

It  has  been  known  for  some  time  that  there  exist  in  higher  plants 
corpuscular  elements  called  plastids  or  leucoplastids,  which  also  possess 
a  synthetic  function.  Some,  the  chloro  plastids,  make  the  chlorophyl 


A 

FIG.  10.  —  Formation  of  chloroplasts  in  the  young  leaf  of  barley.  A,  Very  young 
cells  in  which  appear  rod-mitochondria.  B,  Older  cells  in  which  the  rod-mitochondria 
are  transforming  themselves  into  chloroplasts.  C,  Cells  in  which  the  chloroplasts 
are  definitely  constituted. 

which,  by  using  rays  of  light  as  energy,  forms  starch;  others,  the 
amylo  plastids,  confine  themselves  to  forming  starch  from  the  excess 
sugars  found  in  the  cells;  still  others,  the  chromo  plastids,  constitute  the 
pigment  bodies  of  plants  (xanthophyl,  carotins).  It  has  been  recently 
shown  that  plastids  are  nothing  but-  mitochondria  which  have  under- 
gone greater  differentiation  and  specialization  than  those  which,  at  the 
expense  of  ordinary  mitochrondria  derived  from  the  egg,  have  increased 
in  size  (Figs.  10,  n). 

Mitochondria  have  been  found  in  most  protozoa  and  fungi.  In  the 
latter  they  take  part  in  the  formation  of  reserve  products,  especially 
the  metachromatic  corpuscles  of  which  more  will  be  said  later. 

Mitochondria  are  most  highly  developed  in  algae  where  they  give 
origin  to  chloroplastids  as  in  higher  plants,  On  the  other  hand,  in 


ELEMENTS    OF   MICROBIAL   CYTOLOGY 


21 


the  lower  forms,  no  mitochrondria  seem  to  exist,  but  the  chloroplastids 
take  on  certain  special  characteristics.  Instead  of  small  scattered 
corpuscles  is  found  one,  or  occasionally  several,  large  chloroplastids 
filling  most  of  the  cell.  They  are  in  various  shapes — ribbons,  spirals, 
nets,  etoilated  bodies  (Fig.  12),  etc.— but  all  appear  to  be  made  up  of 
a  mitochondrial  substance.  Their  physiological  role  is  much  more 
general  than  in  the  chloroplastids  of  higher  plants.  They  produce 
not  only  the  chlorophyl,  but  other  pigment  bodies,  the  starch  or  para- 
mylum,  metachromatic  corpuscles,  and  globules  of  fat.  Conse- 


I 


S^J 

FIG.  ii.  FIG.  12. 

FIG.  ii. — A  cell  from  the  root  of  a  bean  in  which  the  rod-mitochondria  (ch) 
form  in  the  course  of  their  development  amyloplasts  from  which  (p)  spring  grains 
of  starch  (a). 

FIG.  12. — A,  Euglena  viridis  with  its  star-like  chloroplasts  (chl.)  at  the  center 
of  the  organism,  the  pyr6noid  body  (Py)  surrounded  by  grains  of  paramylum  (Par), 
eye-spot  (0),  contractile  vacuole  (v),  flagellum  (/),  nucleus  (n).  (After  Dangeard.) 
B,  Microglena  punctifera,  with  two  elongated  chromatophores  arranged  longitudinally. 
(After  Stein.) 

quently  the  complex  chloroplastids  of  the  algae  with  their  general 
function  have  been  considered  as  a  special  form  of  chondrium  which, 
instead  of  being  scattered  in  the  cytoplasm  as  a  number  of  small 
structures,  finds  itself  gathered  in  very  compact  masses. 

The  Cyanophycea  are  the  only  microorganisms  in  which  the  chon- 
drium has  not  been  found.  In  the  Cyanophycea  the  chlorophyl  and  the 
blue  pigment  (phycocyanin)  associated  with  it  are  diffused  throughout 
the  cytoplasmic  area  surrounding  the  nucleus.  The  very  primitive 
structure  of  the  algae  explains  to  some  extent  this  absence  of  an  im- 
portant structure  of  the  cell. 


22  MORPHOLOGY   AND    CULTURE    OF   MICROORGANISMS 

Vacuoles. — There  is  always  in  the  cytoplasm  one  (or  several)  rather 
bulky  vesicle  filled  supposedly  with  an  aqueous  solution  of  mineral 
salts  called  a  vacuole.  Vacuoles  play  an  important  part  in  the  ab- 
sorption of  liquids  by  the  cell.  Owing  to  the  mineral  salts  dissolved 
in  the  vacuole-fluid,  the  concentration  of  which  is  ordinarily  higher 
than  that  of  the  surrounding  medium,  the  vacuoles  become  the  center 
of  osmotic  forces  which  consequently  cause  a  part  of  the  ambient 
liquid  to  penetrate  the  cell  and  determine  its  turgescence. 

Very  curious  vacuoles  are  found  in  many  protozoa,  namely,  the 
pulsating  vacuoles  (Figs.  8,  12).  They  are  small  vacuoles  which  expand 
and  contract  rhythmically,  and  which  are  considered  as  excretory  and 
respiratory  organs.  The  water  that  has  entered  the  cell  gathers  in  this 
vacuole  and  is  expelled  as  it  contracts.  Probably  in  crossing  the  body 
this  water  yields  its  oxygen  to  the  cytoplasm  in  order  to  charge  itself 
with  carbonic  acid  and  the  products  of  metabolism. 

Reserve  Products. — The  cytoplasm  encloses  some  structures  differ- 
entiable  by  means  of  certain  stains  or  chemical  reagents  as  granulations, 
but  which  are  not  constituent  elements  of  cytoplasm;  they  come 
from  a  secretion  of  the  cytoplasm,  and  only  under  certain  conditions. 
These  grains  may  be  found  either  in  the  cytoplasmic  substance  itself, 
or  in  the  vacuoles  included  in  the  cytoplasm.  Most  of  these  granules 
are  reserve  products  which  appear  when  nutrition  is  deficient.  Among 
the  reserve  products  most  common  in  microorganisms  are  the  granules 
called  metachromatic  corpuscles  (Fig.  13,  A).  These  bodies,  which 
are  the  object  of  a  special  study  in  connection  with  molds  and  yeasts, 
are  made  up  of  a  substance  the  nature  of  which  is  still  unknown,  and 
are  found  in  nearly  all  fungi,  in  most  algae  and  bacteria,  and  in  many 
protozoa. 

Glycogen  and  paraglycogen  are  equally  well  distributed  in  micro- 
organisms (fungi,  protozoa).  Among  algse,  glycogen  is  found  only 
in  the  Cyanophycea,  but  it  is  elsewhere  replaced  by  starch  or  para- 
mylum  (Fig.  n),  common  products  of  chlorophyllic  assimilation. 

There  are  also  the  protein  substances,  such  as  crystalloids  of 
mucorin  scattered  in  the  Mucorina,  or  the  globules  of  fat  common 
in  all  cells  (Fig.  13,  £). 

Most  of  these  substances  seem  to  result  from  the  activity  of  the 
chondrium  structure.  Recent  investigation  shows  that  the  meta- 
chromatic corpuscles  have  their  rise  among  the  mitochondria,  It 


ELEMENTS   OF   MICROBIAL   CYTOLOGY  23 

has  long  been  known,  on  the  other  hand,  that  the  starch  and  paramylum 
are  always  formed  in  the  chloroplastids. 

MEMBRANE. — The  cell  is  usually  enveloped  in  a  more  or  less  heavy 
membrane,  secreted  by  the  cytoplasm,  which  acts  as  a  protective 
organ  for  the  cell. 

The  presence  of  the  membrane  is  not,  however,  indispensable; 
many  protozoa  do  not  have  it,  and  are  consequently  naked  cells. 
Motility  in  many  microorganisms  is  closely  associated  with  the  mem- 
brane, for  the  movement  of  cytoplasm  and  the  flexibility  of  the  mem- 


FIG.  13. — A,  Metachromatic  corpuscles  (cm)  in  Sarcosporidia,  Sarcocystis  tenella. 
(After  Erdmann.}  B,  Fat  globules  (g)  in  Trypanosoma  rotatorium.  (Ajter  Doflein.} 

brane  are  essential  factors.  Cells  as  a  rule  have  a  membrane  of 
different  degrees  of  thickness  and  composition.  It  may  be  albuminoid 
or  chitinous  (Infusoria],  or  it  may  be  made  up  of  carbohydrates,  as 
cellulose,  pectose,  and  callose  (algae,  fungi).  Bacteria  always  have  a 
membrane,  but  its  nature  has  not  yet  been  definitely  determined. 
Often  the  cell  membrane  is  able  to  thicken  noticeably,  and  thus  protect 
the  cell  from  influences  of  environment;  the  cell  may  then  be  regarded  as 
transformed  into  a  cyst  which  passes  into  a  state  of  sluggish  existence. 
Encystment  is  frequent  with  protozoa,  and  is  produced  when  the 
environment  becomes  unfavorable  (Fig.  14,  A). 

The  external  layer  of  the  membrane  frequently  undergoes  modi- 
fications, transforming  itself  into  a  mucilaginous  or  gelatinous  sub- 


24  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

stance  as  we  see  in  many  Cyanophycecz,  in  bacteria  surrounded  by 
capsules,  and  in  zooglea.  The  membrane  then  becomes  extremely 
thick  (Fig.  14,  B). 

LOCOMOTIVE  STRUCTURE. — Most  algae  and  fungi  cannot  move. 
Many  bacteria  and  all  protozoa  have  more  or  less  perfected  locomotive 
structure. 

The  CyanophycecB  and  many  bacteria,  although  without  loco- 
motive organs,  present  nevertheless  oscillatory  movements  which  seem 
due  to  a  general  movement  of  the  cytoplasm  translated  exteriorly 
because  of  the  flexibility  of  their  membrane.  With  these  exceptions, 
movement  is  effected  by  means  of  a  locomotive  structure. 

This  structure  is  found  in  its  simplest 
.form  in  the  pseudopodia  of  the  amosba. 
The  naked  cell  of  the  amosba  pushes  out 
pseudopods,  simple  expansions  of  the  ecto- 
plasm arising  at  any  part  of  the  body, 
which  take  various  shapes,  and  reenter  the 
body  without  leaving  the  least  trace  of  their 
existence.  It  is  a  result  of  motility  of  the 
cytoplasm,  one  of  its  essential  properties, 

£  shown  here  exteriorly  because  of  the  absence 
FIG.     14. — A,     Cyst     of  f         »  i               •, 
Amcebalimax.    (After  Dan-  of  a  cellular  membrane. 
geard.}    J5,  Thread  of  nostoc  The  locomotive  structure  is  more  corn- 
surrounded  by  a  thick  muci-  i         .          ., 
laginous  case.  Plex  -m    other    protozoa;    the    pseudopod 

is  replaced  by  contractile  appendages — 
flagella,  or  vibratile  cilia. 

The  flagellum  is  a  contractile  appendage  of  definite  shape  and 
position  which  draws  the  body  after  it  by  means  of  waving  movements. 
It  is  found  on  bacteria  and  flagellates. 

The  organ  of  locomotion  of  bacteria  is  still  little  known  (Fig.  15). 
It  consists  of  a  certain  number  of  contractile  appendages  placed  at 
one  end  of  the  cell,  or  at  both,  or  sometimes  distributed  over  the  whole 
body.  These  appendages,  which  may  be  called  vibrating  appendages, 
have  the  characteristics  of  flagella.  Their  existence,  for  a  long  time 
doubted,  is  now  well  established. 

The  locomotive  structure  of  the  Flagellata  is  much  better  known. 
It  is  characterized  by  one  or  more  flagella  inserted  in  the  anterior 
extremity  of  the  cell.  In  case  of  more,  one  frequently  folds  back 


ELEMENTS    OF   MICROBIAL   CYTOLOGY  25 

toward  the  posterior  end.  In  the  lateral  region  of  the  cell  it  unites 
with  a  contractile  membrane,  the  undulating  membrane,  running  in 
spiral  form  along  the  length  of  the  body,  of  which  it  is  the  free  end. 
Flagella  are  made  up  of  one  or  more  elastic  fibers,  surrounded  by  a 
thin  cytoplasmic  sheath. 

The  vibrating  cilia  are  also  contractile  appendages,  differing  from 
the  flagella  only  in  their  smaller  size.  They  cover  the  whole  body 
of  the  cell,  as  in  the  case  of  infusoria,  enabling  them  to  move  about 
very  easily  in  liquids.  This  interpretation  is  not  concurred  in  by  all 
investigators. 

Certain  facts  lead  us  to  believe  that  flagella  are  only  transformed 
pseudopods  in  which  the  cytoplasmic  structure  has  changed  and  at  the 
same  time  the  kind  of  movement.  Thread- 
like pseudopods  are  found  with  a  rapid 
rhythmic  movement  which  may  serve  as 
intermediate  forms.  Be  that  as  it  may,  the 
method  of  forming  these  organs  is  of  special 
interest.  Apparently  they  are  formed  under 

the  influence  and  at  the  expense  of  the  cen-  FIG.  15.— Organs  of  loco - 
tri'ni~  motion  in  bacteria.  A,  B. 

subtilis.       (After     Fischer) 
In  the  Flagellata  the  flagellum  is  always    B,   Microspira   comma. 

inserted  in  the  centriole  or  in  a  similar  organ    Wter  Fischer  and  Migula.) 

.  C,  Spirillum  rubrum. 

which  appears  to  issue  from  the  centriole. 

It  is  not  rare  to  find  in  cellular  division  some  cells  in  which  the  nucleus 
is  dividing  with  a  centriole  at  each  of  its  poles.  Each  serves  as  a  point 
of  insertion  for  a  flagellum  (Fig.  16,  A,  D,  E). 

According  to  recent  works,  the  flagellum  is  formed  in  general 
in  one  of  two  somewhat  different  methods. 

In  the  first  case,  the  centriole  divides  itself  by  an  elongation,  followed 
by  a  contraction  into  two  centrioles  which  remain  united  to  each 
other  by  means  of  a  fine  thread,  the  centrodesmose.  The  centrodesmose 
then  elongates  and  is  transformed  into  a  flagellum. 

In  the  second  case,  the  centriole  divides  itself  a  first  time  just  as 
in  the  preceding  case,  but  the  centriole  farthest  from  the  nucleus  im- 
mediately undergoes  a  second  division,  thus  making  three  centrioles. 
The  one  nearest  the  nucleus  remains  a  centriole  during  nuclear  division. 
The  centriole  situated  somewhat  farther  from  the  nucleus  becomes  the 
point  of  insertion  for  the  flagellum,  and  is  called  the  blepharoplast  or  basal 


26  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

grain.  The  centriole  is  united  to  the  blepharoplast  by  a  centrodesmose, 
the  rhizoplast,  which  is  often  absorbed.  Finally,  the  last  centriole 
situated  beyond  the  blepharoplast  about  equally  distant,  also  unites 
with  this  cell-organ  by  a  centrodesmose  and,  by  approaching  the 
extremity  of  the  cell,  causes  the  elongation  of  the  centrodesmose  which 
transforms  itself  into  a  flagellum. 

In  the  infusoria  the  vibratile  cilia  insert  themselves  in  the  ectoplasm 
and  pass  through  the  cuticle  to  reach  the  exterior.     At  the  point  of 


L  ^'iBBI       ;<end 

FIG.  16.  FIG.  17. 

FIG.  1 6. — A,  Spongomonas  uvella.  The  nucleus  is  undergoing  mi  to  tic  division. 
Two  centrioles,  each  at  the  base  of  a  flagellum,  are  located  at  the  two  extremes  of 
the  spindle.  (After  Hartmann  and  Chagas.) 

B,  M.onas  termo.  The  cell  lies  in  repose;  a  centriole  (a)  lies  at  the  base  of  the 
flagellum;  in  (C)  there  are  two  centrioles,  in  (D)  the  two  centrioles  occupy  the  two 
poles  of  the  nucleus  during  the  process  of  mitosis;  in  (£)  exists  the  final  nuclear 
division.  (After  Martin.) 

FIG.  17. — Fragments  of  the  peripheral  portion  of  Prorodon  Ieres  (infusorian) 
with  vibratile  cilia  and  their  basal  corpuscles,  (ect)  Ectoplasm;  (end)  endoplasm; 
(tr)  trichocysts.  (After  Maier  and  Gurwitch.) 


insertion  of  each  of  these  cilia  is  a  small  chromatic  corpuscle  or  basal 
grain,  a  trichocyst,  also  supposed  to  arise  from  a  repeated  division  of 
the  centriole  (Fig.  17). 

The  centriole  which,  as  we  shall  see  later,  seems  to  be  a  motor 
organ  associated  with  the  internal  cytoplasmic  movements  during 
cellular  division,  appears  also  to  be  connected  with  the  external  move- 
ment of  the  cell. 


ELEMENTS   OF  MICROBIAL   CYTOLOGY 


REPRODUCTION  OF  THE  CELL 

VARIOUS  PROCESSES  OF  REPRODUCTION. — Reproduction  of  microbes 
affected  by  various  processes;  the  cell  may  reproduce  itself  by  trans- 
verse or  longitudinal  fission,  binary  division,  schizogony  (bacteria, 
flagellata,  molds,  Figs.  6,  A;  18;  20,  A).  This  is  by  far  the  most  fre- 
quent. It  sometimes,  however,  divides  itself  by  budding,  gemmula- 
tion  (Yeast,  Fig.  3);  that  is,  by  the  formation  of  a  small  protuberance 
which  separates  itself  from  the  mother  cell  as  a  small  daughter  cell 
which,  once  free,  grows  slowly  to  maturity. 

Finally,  a  last  process  and  a  very  frequent  one  is  the  formation  of 
internal  spores,  or  sporogony  (Fig.  19).  The  nucleus  undergoes  a 


FIG. 1 8. — Schizogony  in  Amoeba 
polypodia  with  amitotic  division 
of  the  nucleus.  (After  Schulze 
and  Lange.} 


FIG.  19. — Sporogony.  A,  Formation 
of  spores  in  Saccharomyces  cerevisia.  B, 
Formation  of  spores  in  B.  mycoides.  (After 
Guilliermond.)  C.  Formation  of  spores  in 
Leucocytozoon  lovati.  (After  Fanlham.) 


certain  number  of  divisions,  and  the  cytoplasm  divides  itself  inside  the 
cell  in  as  many  small  cells  as  there  are  nuclei.  These  cells  become 
spores  and  are  set  free  by  a  rupture  in  the  wall  of  the  mother  cell. 
Sometimes  all  the  cytoplasm  of  the  mother  cell  divides  into  spores,  and 
sometimes  only  a  part  of  the  cytoplasm  is  used,  the  rest  epiplasm 
serving  as  nourishment  to  the  spores  during  their  growth. 

Whatever  the  means  by  which  the  cell  reproduces  itself,  cyto- 
plasmic  changes  and  nuclear  changes  take  place  at  the  same  time. 
The  most  important  of  the  cytoplasmic  changes  is  the  distribution 
of  the  chondrium  structure  between  two  daughter  cells,  often  preced- 
ing the  division  of  this  cytoplasmic  structure  (Fig.  9). 


28  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

The  nuclear  phenomena  are  much  more  important,  and  better 
known.  The  nucleus  divides  in  order. to  furnish  each  daughter  cell 
with  a  nucleus  containing  the  same  amount  of  chromatin. 

NUCLEAR  DIVISION. — Nuclear  division  may  occur  in  one  of  two 
ways,  one  very  complex,  (i)  the  indirect  mode,  karyokinesis  or  mitosis; 
the  other  very  simple,  (2)  the  direct  mode,  or  amitosis. 

Indirect  Division,  Karyokinesis,  or  Mitosis. — We  shall  begin  with 
the  indirect  mode  which  is  by  far  the  more  common,  using  as  an  example 
a  Heliozoon,  the  Acanthocystis  aculeata  (Fig.  20,  A).  The  nucleus  of 
this  protozoon  at  rest  contains  a  large  karyosome  of  a  spongy  structure, 
and  a  chromatic  network.  Outside  the  karyosome  in  the  nuclear 
vesicle  is  a  centriole  surrounded  by  a  hyaline  zone,  the  archoplasm 
(Fig.  20,  A,  a). 

Mitosis  may  be  divided  into  four  steps  or  phases. 

The  first  phase  or  prophase  begins  by  the  emigration  of  the  centriole 
from  the  nucleus  outside  of  which  it  surrounds  itself  by  cytoplasmic 
irradiations,  making  a  star-like  body,  called  the  aster  (Fig.  20,  A,  b). 
Following  this,  the  karyosome  dissolves  in  the  nucleoplasm,  supposedly 
conveying  material  to  the  chromatic  network  which  enriches  itself 
noticeably  in  chromatin.  The  chromatic  network  then  relaxes,  thickens 
and  transforms  itself  into  a  more  or  less  spiral  cluster,  the  spireme 
(Fig.  20,  A ,  c) .  At  the  same  time  the  centriole  divides  into  two  centrioles, 
each  surrounded  by  an  aster  (Fig.  20,  A,  c}.  Soon  these  centrioles  place 
themselves  at  the  two  opposite  poles  of  the  nucleus  (Fig.  20,  A,  d),  while 
the  spireme  breaks  itself  up  into  a  definite  number  of  chromatic  sec- 
tions, the  chromosomes.  While  this  is  taking  place,  the  nuclear  mem- 
brane dissolves  itself  into  a  series  of  cytoplasmic  fibrils,  the  achromatic 
spindle,  resistant  to  nuclear  stains.  They  appear  in  the  middle  of 
the  nucleus  and  converge  at  each  end  to  the  centrioles  (Fig.  20,  A,  d, 
c).  The  chromosomes  group  themselves  in  the  center  of  the  spindle 
as  the  equatorial  plate  (Fig.  20,  A,  e),  the  formation  of  which  completes 
the  prophase.  Each  of  the  chromosomes  is  attached  to  one  of  the 
fibrils  which  make  up  the  achromatic  spindle. 

The  second  phase  or  metaphase  consists  of  the  longitudinal  di- 
vision of  the  chromosomes  each  of  which  divides  itself  into  two  equal 
chromosomes. 

In  the  third  phase  or  anaphase  the  chromosomes  equally  divided 


ELEMENTS    OF   MICROBIAL   CYTOLOGY 


move  to  the  two  poles  where  they  make  two  polar  plates.     The  cen- 

trioles  located  here  seem  to  have  some  attraction  for  the  chromosomes. 

Finally  comes  the  telophase  or  phase  of  reconstitution  of  the  two 

nuclei  which  terminates  the  process.     In  this  phase,  the  chromosomes 


B 


•  4  •]    * 

*     f     5 


FIG.  20. — Karyokinesis  (metamitosis) .  A,  Acanthocystis  aculeata;  (a)  nucleus 
in  state  of  repose  with  an  intranuclear  centriole;  (b)  (prophase)  the  centriole  moves 
to  the  periphery  and  out  of  the  nucleus  and  forms  an  aster  (After  Hertwig) ;  (c)  the 
division  of  the  centriole  and  spireme;  (d)  the  formation  of  the  equatorial  plates  and 
the  achromatic  spindle;  (e)  equatorial  plates;  (/)  anaphase;  (g)  telophase.  (After 
Schaudinn.)  B,  In  Coleosporium  senecionis  (Uredineae).  (a)  Nucleus  at  rest  with 
its  centriole  extranuclear;  (6)  formation  of  chromosomes;  (c)  equatorial  plate;  (d) 
metaphase;  (e)  anaphase;  (/)  (g)  (i)  telophase.  (After  Madame  Moreau.) 

form  a  spiral  chromatic  cluster  making  a  spireme  at  each  of  the  poles 
(dispireme  stage,  Fig.  20,  A, g);  each  of  the  spiremes  is  then  surrounded 


30  MORPHOLOGY  AND   CULTURE   OF   MICROORGANISMS 

by  a  nuclear  membrane  in  which  is  included  the  centriole.  Thus  the 
two  nuclei  are  formed  in  which  a  nucleolus  soon  appears.  Mean- 
while the  cell  has  elongated,  become  constricted  in  the  center,  and 
finally  broken  into  two  cells  (Fig.  20,  B,  f,  g,  f).  The  achromatic 
spindle  completely  disappears. 

This  method  of  division  represents  the  typical  method  of  karyo- 
kinesis,  that  which  is  observed  in  higher  organisms  with  the  single 
difference  that  the  centriole  is  intranuclear,  whereas  in  the  cells  of 
higher  organisms  it  is  ordinarily  outside  the  nucleus  in  contact  with  the 
nuclear  membrane.  An  analogous  mitosis  is  found  in  the  Uredinea 
(Fig.  20,  B,  a-i),  except  that  the  centriole  is  here  found  to  be  extra- 
nuclear  (Fig.  20,  B,  a),  the  asters  are  lacking,  and  the  nucleolus  persists 
to  the  end  of  mitosis  expelled  in  the  cytoplasm.  The  physiological 
significance  of  the  nucleolus  in  this  case  is  not  known.  This  method  of 
division  is  seen  in  certain  molds  and  higher  protozoa,  and  is  called 
metamitosis  or  perfect  mitosis. 

Summing  up,  mitosis  is  a  process  functioning  to  make  an  absolutely 
equal  division  of  the  chromatin  between  the  two  nuclei.  This  dis- 
tribution is  performed  by  the  breaking  up  of  a  spireme  into  a  definite 
number  of  chromosomes,  a  number  varying  according  to  the  species 
but  always  constant  for  any  single  species,  and  then  by  a  longitudinal 
division  of  the  latter.  The  centrioles  seem  to  play  an  important  role 
in  this  phenomenon,  in  directing  it,  and  in  attracting  the  chromosomes 
once  divided  toward  the  poles  of  the  cell  where  the  nuclei  are  formed. 

It  is  not  necessary  to  conclude  that  the  processes  of  mitosis  are 
as  complex  as  in  other  microorganisms.  Relatively  simple  in  the 
lower  forms,  mitosis  becomes  complicated  as  it  climbs  the  ladder, 
gaining  the  characteristics  of  metamitosis  only  in  the  most  advanced 
forms. 

The  simplest  case  is  found  in  the  Cyanophycea  (Fig.  6).  Here 
cellular  division  begins  by  the  outline  of  the  transverse  partition 
which  appears  in  the  form  of  a  peripheral  ring.  At  the  same  time 
the  chromatic  network  takes  a  definite  arrangement;  its  filaments 
arrange  themselves  parallel  to  the  longitudinal  axis  of  the  cell,  thus 
giving  this  division  the  appearance  of  a  mitotic  division.  The  outline 
of  the  partition  extends  little  by  little  toward  the  middle  of  the  cell, 
leaving  open  only  a  small  spherical  space  in  its  center  to  which  the 
fibers  of  the  network  then  contract,  and  the  nucleus  takes  the  form  of 


ELEMENTS    OF   MICROBIAL   CYTOLOGY 


31 


• 


D 


(B    (I 


9 


• 

e 


FIG.  21. — Protomitosis.  A,  In  Amceba  mucicola.  (a)  Nucleus  at  rest;  (b) 
beginning  of  prophase;  (c)  division  karyosome;  (d)  division  of  centriole;  (e)  (/) 
equatorial  plate;  (g)  metaphase;  (i)  (k)  telophase.  (After  Chatton.)  B,  In  Amoeba 
froschi.  (After  Nagler.)  C,  In  Euglena  splendent.  (After  Danteard.)  D,  In 
4 mxba  diplomitotica.  (After  Beaurepaire  Arago.)  t 


32  MORPHOLOGY   AND    CULTURE    OF   MICROORGANISMS 

a  dumb-bell.  Soon  the  partition  stops  completely,  the  filaments  of 
the  contracted  part  of  the  nucleus  break  up  and  the  two  daughter  cells 
appear  separated  by  a  partition.  The  two  nuclei  whose  filaments  have 
been  sectioned  by  the  partition  are  not  slow  in  recovering  their  in- 
tegrity (Fig.  6,  b). 

We  find  in  the  Amoeba  mucicola  (Fig.  21,  A)  a  much  more  char- 
acteristic mitosis,  though  more  primitive.  The  nucleus  of  this  amoeba 
when  at  rest  is  made  up  of  a  nuclear  fluid  surrounded  by  a  membrane 
in  which  are  a  large  karyosome  and  some  small  grains  of  chromatin 
localized  on  the  periphery  (Fig.  21,  A).  In  the  center  of  the  karyosome 
is  a  small  chromophilic  centriole.  The  prophase  begins  by  the  elonga- 
tion of  the  karyosome  to  a  rod-shaped  body  (Fig.  21,  A,  b)  which  then 
transforms  itself  into  a  dumb-bell  (Fig.  21,  A,  c).  The  centriole 
also  elongates  and  becomes  constricted  in  the  center  (Fig.  21,  A,  d). 
At  the  same  time  an  achromatic  spindle  appears  all  about  the  con- 
stricted region  of  the  karyosome  in  the  middle  of  which  the  grains  of 
chromatin  arrange  themselves  peripherally  to  form  an  equatorial 
plate,  but  there_is  no  differentiation  of  this  chromatin  into  two 
chromosomes  (Fig.  21,  A,  c,  d).  In  the  metaphase  the  karyosome 
and  the  centriole  divide  into  two  polar  masses  (Fig.  21,  A,  e,  /),  the 
equatorial  plate  separates  into  two  plates  which,  in  the  anaphase, 
emigrate  to  the  poles  (Fig.  21,  A,  g)  drawn  by  the  centrioles.  In  the 
telophase  the  spindle  elongates,  disappears,  and  the  two  nuclei  are 
formed  at  the  poles  (Fig.  21,  A,  i,  k).  The  nuclear  membrane  exists 
during  the  entire  phenomenon. 

In  other  microorganisms  (Amoeba,  Flagellata,  Euglena)  is  found  a 
similar  mitosis  except  that  the  chromatin  distributed  in  the  resting 
nucleus  as  a  network  or  as  rod-shaped  bodies  forms  an  equatorial  plate 
made  up  of  true  chromosomes  (Fig.  21,  B,  C). 

Another  form  of  mitosis,  promitosis,  is  characterized  by  the  fact 
that  the  centriole  is  included  in  the  karyosome,  by  the  persistence  of 
the  nuclear  membrane,  and  by  the  simultaneous  division  in  the  meta- 
phase of  the  karyosome  and  of  the  chromatin  gathered  in  an  equatorial 
plate. 

Between  promitosis  and  metamitosis  are  a  series  of  intermediate 
forms.  In  the  Pelomyxa  palustris,  for  example,  the  centriole  while 
remaining  intranuclear  is  able  to  separate  itself  from  the  karyosome 
(Fig.  22,  A}  a).  The  prophase  here  begins  with  the  usual  division  of 


ELEMENTS    OF   MICROBIAL   CYTOLOGY 


33 


the  centriole  (Fig.  22,  A,  b,  c),  and  the  two  resulting  centriole- threads 
pass  to  the  extremities  of  the  achromatic  spindle,  while  the  karyosome 
cooperates  in  the  formation  of  the  chromosomes  (Fig.  22,  A,  d,  e). 

In  other  cases  (various  fungi,  Gregarince,  etc.),  the  centriole  be- 
comes extranuclear,  and  the  karyosome  acts  as  a  true  nucleolus  (Fig. 


A 


u 


e 


FIG.  22. — Mesomitosis.  A,  In  Pelomyxa  palustris.  (a)  Nucleus  at  rest;  (&) 
— (e)  division  of  centriole;  (/)  (g)  equatorial  plate;  (h)  anaphase.  (After  Bolt.} 
B,  In  Urospora  lagidis  (Gregarina).  (a)  Nucleus  with  extranuclear  centriole  and 
aster;  (b)  the  centriole  is  divided  and  the  spireme  is  fornied;  (c)  spireme;  (d)  equa- 
torial plate;  (e)  anaphase.  (After  Brasil.)  C,  In  the  ascus  of  Galactima  succosa 
(Ascomycete).  (a)  Equatorial  plate;  (b}  anaphase;  (c)  telophase. 

22).  Sometimes  it  dissolves  at  the  beginning  of  mitosis,  seeming  to 
aid  the  development  of  the  chromatin  of  the  spireme,  and  sometimes 
it  persists  during  the  entire  process  and  is  expelled  in  the  cytoplasm 
at  the  end  of  the  phenomenon  without  any  known  function.  The 


34  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

name  of  mesomitosis  has  been  given  to  all  the  mitoses  which  distinguish 
themselves  from  promitosis  by  the  persistence  of  a  nuclear  membrane 
throughout  the  phenomenon. 

Direct  Division  or  Amitosis. — This  consists  simply  of  an  elongation 
of  the  nucleus  followed  by  a  median  constriction,  then  by  a  rupture  of 
this  constricted  part  without  an  equal  division  of  the  chromatin  be- 
tween the  two  nuclei  which  often  are  not  the  same  size.  It  is  a  simple 
breaking  up  of  the  nucleus.  Amitosis,  then,  does  not  necessarily  in- 
sure the  equal  distribution  of  chromatin  between  the  two  nuclei. 
This  rare  process  is  found  in  higher  organisms  only  in  old  cells  that  are 
degenerating,  or  in  diseased  cells.  Although  for  a  long  time  it  was 
thought  to  be  a  primitive  phenomenon,  it  is  now  considered  to  be 
degenerative.  We  see,  however,  in  certain  Amoeba  and  Mycetozoa 
the  karyosome  enclosing  all  the  chromatin  divides  itself  into  two  equal 


/  *\ 

uO 


FIG.  23. — Conjugation  in  Schizosaccharomyces  octosporus.     (a)   Two  gametes  in 
the  process  of  fusion;  (b)  (c)  nuclear  fusion. 


bodies,  showing  the  characteristics  of  a  very  primitive  mitosis  (Fig.  18). 
Amitosis  seems  to  exist  normally  in  yeasts  and  in  certain  molds.  In 
the  yeasts,  for  example,  the  nucleus  divides  by  amitosis  in  the  course 
of  budding  (Fig.  3),  and  mitosis  is  found  only  in  the  course  of  sporu- 
lation. 

SEXUAL  CHANGES. — In  most  microorganisms  at  certain  times  during 
their  existence  occur  sexual  changes,  or  fertilization,  which  seem  to  give 
them  a  new  strength.  It  is  followed  by  a  period  of  very  active  re- 
production, whence  the  name  of  sexual  reproduction  given  to  these 
changes.  This  consists  essentially  in  the  fusion  of  two  equal 
isogamous  (isogamy)  or  unequal,  anisogamous  (heterogamy)  cells  or 
gametes.  In  the  latter  case,  the  male  is  small  and  active,  and  the 
female  large  and  passive.  The  fusion  between  the  two  cytoplasms 
and  the  two  nuclei  takes  place  at  the  same  time  (Fig.  23). 

If  nuclear  fusion  were  not  compensated  by  an  elimination  of 
chromatin,  the  nucleus  would  increase  in  this  substance  at  each  fertili- 


. 


ELEMENTS    OF   MICROBIAL   CYTOLOGY  35 


tion.  But  this  change  is  succeeded  immediately  in  protozoa  by  a 
common  process  called  chromatic  reduction.  The  chromosomes 
in  the  course  of  the  divisions  which  precede  the  formation  of  the 
gametes  reduce  themselves  to  half  by  a  complex  process  which  it 
would  be  superfluous  to  describe  here.  The  same  chromatic  reduction 
takes  place  in  the  fungi  and  algae,  but  this  does  not  always  precede 
fertilization.  It  may  follow  it  immediately  as  in  the  yeasts  where  it 
seems  to  produce  itself  during  the  nuclear  divisions  in  the  ascus.  It 
may  also  occur  during  other  stages  of  development. 


CHAPTER  II* 

MOLDS 
FUNGI  IN  GENERAL 

Certain  fungi  commonly  designated  molds  are  constantly  met  in 
microbiological  studies.  They  are  found  as  contaminating  colonies  in 
microbial  cultures.  They  are  agents  together  with  other  microorgan- 
isms in  the  processes  of  fermentation  and  decay  in  the  soil,  and  in  the 
spoilage  of  food-stuffs.  Certain  of  these  forms  approach  the  structure 
and  habits  of  other  microorganisms  very  closely.  Members  of  these 
border  groups  have  been  sometimes  described  as  bacteria,  some- 
times as  fungi;  among  them  the  Actinomycetes  have  been  principally 
studied  by  bacteriologists,  but  recently  Drechsler  has  succeeded  in 
describing  their  fruiting  forms  in  terms  which  leave  no  doubt  that 
they  are  a  fungous  group.  In  describing  microorganisms,  cultural 
reactions  have  been  largely  used  in  characterizing  the  bacteria  and 
related  organisms;  morphology  has  been  the  basis  of  nearly  all  descrip- 
tions of  the  mycelial  fungi. 

With  some  exceptions,  there  is,  among  the  cells  of  the  true  fungi, 
a  differentiation  of  function  into  vegetative  or  assimilative  cells  and  re- 
productive cells.  The  fungous  body  is  usually  composed  of  threads 
(technically  called  hypha,  singular,  hypha).  These  hyphce  usually 
branch  in  more  or  less  complex  manner  forming  networks  or  webs, 
collectively  called  mycelium.  Hyphae  may  be  one-celled  or  composed  of 
many  cells  placed  end  to  end  as  shown  by  the  cross  walls,  called  septa, 
seen  in  them.  These  threads  grow  either  by  the  formation  of  new  cells 
at  the  growing  tips  (called  apical  growth)  or  by  the  division  of  cells  in 
the  hypha  (intercalary  growth).  The  fungous  cells  rarely  divide  in 
three  planes  to  produce  solid  masses  of  cells.  Both  vegetative  and 
reproductive  masses  are  formed  in  great  variety  from  such  hyphae. 
Often  the  thread-like  character  is  almost  or  quite  obliterated  in  the  ripe 

*  Prepared  by  Charles  Thorn.  A.  Guilliermond  has  furnished  the  sections  on  "  Cytology  of 
Molds  " 

36 


MOLDS  37 

masses,  which  may  be  fleshy,  woody,  carbonaceous,  leathery  and 
even  horn-like  in  texture,  as  seen  especially  in  the  mushrooms,  bracket- 
fungi,  etc.,  but  even  in  such  cases  the  early  stages  show  the  structures 
to  originate  from  masses  of  fungous  threads. 

The  formation  of  differentiated  reproductive  cells  is,  in  general, 
characteristic  of  the  fungi.  The  method  of  reproduction  presents  great 
variety.  In  the  simplest  forms,  the  reproductive  cells  are  scarcely,  if 
at  all,  distinguishable  from  the  vegetative  cells.  In  some  species  whole 
hyphae  break  up  so  that  each  cell  forms  the  starting-point  of  a  new 
colony.  Other  forms  develop  special  branches  bearing  reproductive 
cells.  From  these  it  is  but  a  step  to  the  production  of  fruiting  branches, 
characteristic  in  form,  called  conidiophores,  bearing  cells  markedly 
specialized  as  reproductive  by  form  and  frequently  also  by  color, 
called  conidia.  These  conidia  are  entirely  asexual  in  origin  and  capable 
of  growing  directly  into  new  colonies,  although  in  many  cases  they  are 
provided  with  resistant  walls  which  enable  them  to  live  for  long  periods 
if  conditions  are  unfavorable  to  growth  at  once.  In  other  species, 
specialized  resting  cells  with  resistant  walls  are  formed  to  enable  the 
plant  to  survive  unfavorable  conditions.  These  are  called  chlamydo- 
spores  or  sometimes  cysts.  The  name  gemma  is  sometimes  applied  to 
similar  structures,  preferably  to  such  as  grow  at  once.  The  same  end  is 
reached  in  still  other  groups  by  the  formation  of  sderotia  which  are 
hard  masses  or  balls  of  thick- walled  cells  filled  with  concentrated  food 
materials.  These  sclerotia  are  frequently  distinctive  of  the  species 
producing  them  by  size  and  appearance.  They  vary  from  microscopic 
in  size  to  masses  weighing  many  pounds  and  may  perhaps  in  cases  be 
aborted  fruits.  They  sometimes  resemble  such  sexual  fruiting  bodies. 
Resting  structures  of  either  type,  especially  when  large,  commonly 
produce  typical  spore-bearing  structures  at  once  after  germinating. 
Sexuality  among  the  fungi  has  been  difficult  to  demonstrate  in  some 
groups  with  complex  fruit  bodies.  It  is  certainly  suppressed,  if  not 
entirely  wanting,  in  whole  series  of  cosmopolitan  forms  and  present 
only  in  rare  species  in  other  groups. 

The  systems  of  classification  used  are  largely  based  upon  the  types  of 
sexual  fruit  bodies  produced.  Where  such  fruit  bodies  are  not  known, 
the  method  of  formation  of  the  asexual  spores  furnishes  the  most 
satisfactory  basis  for  grouping.  In  classifying  fungi,  certain  types  of 
spore  formation  are  found  to  be  characteristic  of  particular  groups. 


38  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

Since  within  these  groups  various  accessory  types  of  fruiting  occur,  so 
that  some  species  show  three  or  even  more  forms  of  spores,  that  type 
of  spore  formation  which  is  regarded  as  characteristic  of  the  group  is 
known  as  the  perfect  stage.  If  sexual  fruits  are  found,  these  constitute 
the  perfect  stage  of  the  group;  if  no  such  fruit  is  found,  the  most 
characteristic  asexual  form  is  used. 

Between  the  typical  forms  are  many  gradations  resulting  in  many 
families  whose  relationship  to  one  or  the  other  group  is  difficult  to 
determine.  Probably  the  ancestral  history  (phylogeny)  of  the  fungi, 
if  known,  would  show  several  or  many  lines  of  descent  rather  than  one. 
Many  thousands  of  species  of  fungi  have  been  described,  principally 
in  Latin,  German,  French  and  English.  The  literature  is  widely 
scattered  in  monographs,  reports  and  journals  published  in  various 
languages.  For  the  purposes  of  the  bacteriological  worker,  a  few 
representative  groups  with  some  of  the  significant  species  will  be 
considered  here. 

BACTERIA. — In  the  scheme  of  plant  grouping  presented  (page  13), 
which  is  only  one  of  many  attempts  to  show  relationships,  the  bacteria 
are  placed  with  a  group  of  single-celled  green  or  blue-green  forms  as 
Schizophyta  or  fission-plants  -because  of  reproduction  only  by  the  divi- 
sion of  the  cells.  Recent  work  of  Lohnis,  Cort  and  others  have  opened 
possibilities  of  specialized  spore-production  in  the  bacteria.  Such 
reproductive  bodies,  if  proved  present  and  fully  described,  would 
probably  furnish  a  sound  basis  for  a  scheme  of  relationships  of  the 
bacteria.  At  present  it  is  undecided  whether  they  are  specialized 
from  higher  forms  by  suppression  of  characters  or  represent  a  primitive 
morphology  with  highly  specialized  physiological  relations. 

PHYCOMYCETES. — The  Phy corny cetes  are  called  algal  fungi  because 
they  resemble  certain  groups  of  green  filamentous  forms  in  many 
particulars.  In  this  group  two  general  types  of  sexual  reproduction 
appear — zygospore  formation  and  oospore  formation.  The  first, 
found  in  the  Zygomycetes  represented  by  the  common  mucors,  consists 
of  the  fusion  of  terminal  cells  of  branches  of  the  mycelium  similar  in 
appearance  but  differentiated  in  sex.  As  a  result  of  this  fertilization 
large  thick-walled  resting  cells  are  produced,  called  zygospores,  from  a 
Greek  root  meaning  yoked  (Fig.  33).  In  oospore  formation,  found 
in  the  Oomyceles,  the  conjugating  cells  differ  in  appearance  as  well  as 
in  function.  The  oospore  or  egg-cell  is  large  and  is  rich  in  food 


MOLDS  39 

materials;  the  antheridium  is  much  smaller,  penetrates  and  fertilizes 
the  oospore,  which  afterward  develops  into  a  thick- walled  resting  spore. 
The  very  destructive  downy  mildews  belong  to  this  group. 

ASCOMYCETES. — In  this  great  group  sexuality  was  denied  until  recent 
years,  but  has  been  proved  in  cases  enough  to  establish  a  presumption  of 
more  general  occurrence.  The  characteristic  structure  of  the  group  is 
the  ascus,  a  sac  containing,  when  ripe,  typically  eight  spores,  some- 
times a  less  number  by  the  failure  of  some  to  develop,  sometimes  a  larger 
number,  usually  some  multiple  of  eight.  The  ascus  when  sexuality  is 
known  is  developed  subsequent  to  fertilization,  not  directly  from  an 
egg  cell.  The  group  presents  a  great  variety  of  fruiting  masses  pro- 
duced in  connection  with  the  asci.  The  simplest  forms  are  loose  webs 
of  hyphae  enmeshing  a  few  asci;  other  forms  show  clubs,  cups,  flask 
forms,  crusted  areas,  the  type  of  mass  in  each  case  being  characteristic 
of  the  family,  genus  and  species  represented.  Only  a  few  of  many 
thousands  of  these  forms  are  encountered  in  bacteriological  work. 
One  genus  is,  however,  constantly  found.  The  commonest  species  of 
Aspergillus  produces  bright  yellow,  globose  fruiting  bodies,  called 
perithecia  (singular,  perithecium) ,  filled  with  asci.  These  are  borne  upon 
the  surface  of  the  substratum  and  often  give  a  yellow  color  to  the  colony 
by  their  abundance.  Such  perithecia  consist  of  the  ascogenous  ceils 
and  the  asci  produced  by  them,  about  which  a  more  or  less  completely 
closed  sac  or  wall  has  been  formed,  by  the  development  of  the  sterile 
cells  adjacent  to  the  fruiting  ones. 

BASIDIOMYCETES. — In  the  Basidiomycetcs  there  is  still  further  reduc- 
tion of  the  evidences  of  sexuality. 

In  some  sections  of  this  group  an  essential  sexuality  has  been  corre- 
lated with  the  fusion  of  nuclei  at  stages  of  life  history  characteristic 
for  the  particular  sections  of  the  group.  Such  fusion  seems  to  underlie 
the  development  of  the  typical  faint  body,  although  it  has  only  been 
demonstrated  in  a  small  number  of  species.  The  typical  structure  is 
the  basidiuniy  a  spore-bearing  cell  characteristically  producing  four 
protuberances  called  sterigmata  (singular,  sterigma),  each  bearing  a 
single  spore.  These  basidia  are  grouped  into  many  kinds  of  fruit  bodies 
varying  from  occurrence  here  and  there  upon  a  loose  web  of  hyphae  to 
dense  columnar  areas  covering  the  gills  of  the  mushrooms  or  lining  the 
cavities  of  the  puffballs.  Very  few  of  these  species  are  encountered  in 
bacteriological  studies. 


40  MORPHOLOGY   AND    CULTURE    OF   MICROORGANISMS 

IMPERFECT  FUNGI. — A  very  large  number  of  species  are  known 
which  have  never  been  seen  to  produce  sexual  fruits  or  fruits  character- 
istic of  the  great  groups.  These  are  brought  together  and  described  as 
form-genera  by  their  method  of  asexual  spore  formation.  From  the 
lack  of  the  organs  used  in  classifying  the  other  groups,  these  are  called 
the  Imperfect  Fungi  and  their  grouping  regarded  only  as  temporary, 
a  convenience  for  the  identification  of  materials.  These  include  many 
forms  of  economic  importance,  and  many  of  the  species  most  frequently 
met  in  bacteriological  work.  Sometimes  one  or  a  few  species  of  a  large 
group  produce  the  perfect  form  while  very  many  species  cannot  be 
induced  to  do  so.  Some  of  these  species  undoubtedly  represent  stages 
of  perfect  fungi  whose  perfect  forms  simply  are  not  recognized  as 
connected  with  these;  others  as  in  the  more  common  species  of  Peni- 
cillium  reproduce  for  an  indefinite  number  of  generations  by  conidia. 
Such  species  do  not  appear  to  need  the  perfect  form  and  hence  ap- 
parently have,  in  some  cases,  lost  the  power  to  produce  it.  Genera 
consisting  entirely  of  such  species  are  very  properly  retained  as  form- 
genera  in  the  Imperfect  group. 

As  found  in  nature  all  these  forms  are  parasitic,  saprophytic,  or 
capable  of  both  modes  of  life.  All  depend  more  or  less  completely 
upon  organic  matter  for  nourishment.  Great  diversity  exists,  how- 
ever, in  their  adaptation  to  environment.  Many  of  them  are  not  only 
parasitic  but  so  closely  adapted  to  parasitizing  particular  host-species 
as  not  to  be  found  elsewhere.  Others  attack  several  or  many  species, 
usually  related.  Even  among  saprophytes  many  species  are  found  only 
upon  particular  forms  of  decaying  animal  or  vegetable  matter.  The 
great  economic  importance  of  these  parasitic  and  closely  adapted  sapro- 
phytic species  has  been  recognized  by  the  development  in  recent  years 
of  the  literature  of  plant  pathology  (phytopathology).  These  cannot 
be  considered  in  this  work. 

CYTOLOGY  OF  MOLDS* 

GENERAL  STRUCTURE  OF  MOLDS. — Three  kinds  of  cell-structure 
formation  are  found  in  molds: 

i.  Some,  belonging  to  the  Phycomycetes,  show  no  cross- walls;  they 
have  a  much  branched,  felted  mycelium,  but  in  the  early  stages  there 

*  Prepared  by  A.  Guilliermond. 


MOLDS 


are  no  true  transverse  septa.  Septa  appear  in  many  forms  only  when 
fruiting  begins,  but  in  the  opinion  of  some  they  merely  separate  the 
living  portions  of  the  mycelium  from  those  in  which  the  cytoplasm  is 
dead  or  degenerating.  The  cytoplasm  in  the  unseptate  mycelium  forms 
one  continuous  mass;  it  contains  a  great  many  nuclei  (Fig.  24,  i  and 

2).     Each  nucleus  with  the  cyto- 

£':•/;...  plasm  surrounding  it,   according 

/>  to    Sachs,  may  be  considered  a 

,;£:,., :.:;       •;-;,  .    physiological    unit    acting    in    a 

-.-.•  ..'•;•  ,r  ...  somewhat  similar  capacity  as  a 

•;3  (;';•;•  2  .,.v'  cell,  or  may  be  designated  as  an 


FIG.  24.  FIG.  25. 

FIG.  24. — i,  Part  of  the  mycelium  of  Thamnidium  elegans  (Mucor).  2,  Ex- 
tremity ^of  a  filament  of  Mucor  circinetto'ides  showing  three  swellings  about  to  form 
sporangia.  3,  A  spore  of  the  same  mold.  4.  Yeast  forms  from  the  same  mold. 
(After  Leger.) 

FIG.  25. — i,  Mycelial  filament  of  Endomyces  magnusii.  2,  Extremity  of  a 
filament  of  the  same  mold  in  the  process  of  growth,  with  a  dividing  nucleus.  3  and 
4,  Filaments  of  Endomyces  fibuliger.  In  4,  metachromatic  corpuscles  are  seen 
in  the  vacuoles.  5,  Filament  on  the  way  to  increase,  from  the  same  mold,  the 
nucleus  dividing. 

energid.  This  view  is  not  held  by  all  observers,  however.  Con- 
sidered thus,  the  mycelium  represents  the  collection  of  a  great  many 
indistinct  cells  which  are  not  separated  by  walls.  The  Mucorinea,  for 
example,  belong  to  this  structural  type. 

2.  Other  fungi,  especially  among  the  Ascomycetes,  have  a  septate 
mycelium,  but  one  in  which  the  transverse  septa  do  not  restrict  cellular 


42  MORPHOLOGY   AND    CULTURE    OF   MICROORGANISMS 

functions  as  true  cells.  It  consists  of  compartments  containing  a 
variable  number  of  nuclei  called  coenocytes  (Fig.  25,1).  Each  compart- 
ment may  be  considered,  not  as  a  true  cell,  but  as  a  colony  of  rudi- 
mentary cells,  energids. 

3.  Still  other  molds  have  a  mycelium  consisting  of  true  cells  with 
a  single  nucleus,  as  for  example  Endomyces  fibuliger  (Fig.  25,  3  and  4) 
and  Endomyces  decipiens. 

There  are,  moreover,  molds  which  show  both  these  last  two  struc- 
tural types,  with  transitional  forms  between  the  two.  For  instance, 
in  Endomyces  magnusii,  the  mycelium,  ordinarily  consisting  of  areas, 
each  containing  many  energids,  can  in  some  parts  progress  to  a 
uninuclear  cellular  structure. 

The  conidia  or  spores  of  many  molds  may  have  either  one  or 
many  nuclei,  according  to  the  species.  The  spores  of  the  Mucorinea, 
for  example,  always  have  many  nuclei  (Fig.  24,  3) ;  on  the  contrary, 
the  ascospores  of  the  Ascomycetes,  the  conidia  of  PenicilUum  and 
Aspergillus,  contain  generally  but  a  single  nucleus. 

The  yeast  forms  which  result  from  the  budding  of  the  mycelium  in 
some  molds,  most  frequently  have  a  single  nucleus  (Fig.  24,  4) ;  how- 
ever, in  some,  Dematium,  are  sometimes  found  yeast-forms  containing 
several  nuclei.  The  yeast-forms  of  the  Mucorinea,  which  are  not  other- 
wise very  typical  forms,  are  always  multinuclear. 

To  whichever  of  these  three  structural  forms  a  mold  belongs,  it 
always  represents  some  similar  constitutional  elements  which  we  will 
now  consider. 

CYTOPLASM. — The  cytoplasm  is  a  semi-fluid  mass,  somewhat  dense, 
sometimes  homogeneous  and  containing  a  more  or  less  considerable 
number  of  vacuoles.  Certain  methods  of  fixing  and  staining  have 
recently  made  possible  a  demonstration,  in  the  cytoplasm  of  the 
most  diverse  molds,  of  the  presence  of  a  chondrium,  very  clear  and 
always  splendidly  exhibited.  This  consists  mostly  of  fine  rod- 
mitochondria,  very  long  and  flexible,  generally  lying  parallel  with 
the  longitudinal  axis  of  the  cell  (Fig.  26).  Sometimes  also  it  contains 
granular  mitochondria. 

The  cytoplasm  also  has  reserve  products,  of  which  we  shall  speak 
later. 

NUCLEI. — The  nuclei  show  a  differentiated  structure  which  is 
sometimes  difficult  to  demonstrate.  They  consist  of  a  nuclear 


MOLDS 


43 


membrane,  a  hyaline  nucleoplasm,  a  large  nucleolus  and  a  chromatic 
network.  The  last  is  sometimes  indistinct,  and  it  frequently  happens 
that  the  nucleus  appears  to  contain  only  a  nucleolus;  but  a  very 
careful  examination  always  reveals  the  network  (Fig.  25,  3  and  4). 

The  division  of  the  nucleus  is  not  always  easy  to  observe.  To  study 
it,  one  must  examine  the  growing  tips  of  the  mycelium.  In  some  cases 
this  consists  in  an  elongation  of  the  nucleus  which  soon  assumes  the 


i  M  * 


6 


FIG.  26. 


FIG.  27. 


FIG.  26. — Various  molds  fixed  and  stained  by  a  special  technic,  showing 
their  chondrium.  i,  Filament  of  Rhizopus  nigr leans  (Mucor).  2-4,  Filaments  of 
Penicillium  glaucum.  5  and  6,  Fragments  of  the  conidial  organ  of  the  same 
mold.  7,  Filament  of  Endomyces  magnusii.  8  and  9,  Oidia  of  the  same  mold. 
In  all  these  molds,  chondrium  is  represented  by  long  filaments,  or  sometimes 
by  small  grains.  The  filaments  often  show  small  vesicles  at  their  crossing. 

FIG.  27. — Nucleus  of  the  Mucor  (1-4),  and  various  stages  of  its  division  (5-8). 
(After  Moreau.} 

form  of  a  very  slender  dumb-bell  which  breaks  apart  at  the  narrow 
portion.  This  is  the  extent  of  an  amitotic  or  direct  division  (Fig.  25, 
2  and  5). 

Karyokinesis  is  usually  seen  only  in  the  organs  of  fructification 
(asci,  basidia,  etc.);  nevertheless,  in  the  mycelium  of  theBasidiomycetes 
and  Mucorinetz,  true  metamitoses  have  been  found.  In  the  Mucorinea 
for  example  (Fig.  27),  the  nucleus  loses  its  membrane  (1-4)  and  gives 


44  MORPHOLOGY  AND   CULTURE   OF  MICROORGANISMS 

rise  to  a  spindle  ending  in  a  centrosome  at  either  extremity,  while  two 
chromosomes  form  the  equatorial  plate  at  the  center  (5).     Each  of 
the  two  chromosomes  divides  and  the  four  resulting  chromosomes  are 
distributed  between  the  two  poles  (6-8)  where 
they  form   the  two  daughter  nuclei  (Moreau). 
/•.\  METACHROMATIC  CORPUSCLES  AND  RESERVE 

V          PRODUCTS. — The    vacuoles    always    contain   a 
'  great  many  shining  granules,  showing  Brownian 

m°ti°n  an(^  caPa°le  of  being  stained  in  the  living 


f*M 

If* 

j£~tvc 

i 


"4  5 

6      H^%^ 


FIG.  28.  FIG.  29. 

FIG.  28. — Dematium  species  stained  by  a  method  permitting  the  differentiation 
of  the  metachromatic  corpuscles,  i,  Filament.  7  and  9,  Yeast  forms.  9,  Yeast 
form  starting  to  bud  from  mycelium.  The  metachromatic  corpuscles  are  situated 
in  the  vacuoles  in  the  form  of  small  grains  joined  in  chains  (6)  or  isolated.  Many 
appear  like  large  granules  (9).  n.  Nucleus,  v.c.  Vacuole  with  metachromatic 
corpuscles. 

FIG.  29. — Various  stages  of  the  development  of  the  ascus  in  Aleuria  cerea. 
i  and  2,  Young  asci  with  their  nucleus  and  many  metachromatic  corpuscles.  3, 
Fragments  of  an  ascus  after  the  second  nuclear  division.  4,  Ascus,  still  young, 
in  which  the  ascospores  are  surrounded  by  metachromatic  corpuscles.  5,  Older 
ascus  in  which  most  of  the  metachromatic  corpuscles  have  been  absorbed  by  the 
ascospores. 

state  by  neutral  red  and  methylene  blue.  These  bodies  have  staining 
qualities  which  permit  them  to  be  easily  characterized.  They  are  stained 
a  violet-red  by  most  of  the  basic  dyes,  aniline  blue  or  violet.  They  also 
take  on  a  very  pronounced  reddish  tinge  with  hematoxylin  (Fig.  28). 
By  reason  of  this  property  of  metachromatism,  they  have  been  called 
metachromatic  corpuscles.  These  bodies,  which  are  very  common  in  the 
Protista,  have  been  found  in  yeasts,  bacteria,  algae  and  protozoa.  The 
chemical  nature  of  the  substance  constituting  them  is  still  unknown, 


MOLDS  45 

but  the  name  metachromatin  is  often  used  for  it.1  Some  authors,  among 
whom  is  Arthur  Meyer,  believe  them  to  consist  of  a  combination  of 
nucleic  acid,  but  this  is  a  mere  supposition. 

On  the  other  hand,  the  role  of  the  metachromatic  corpuscles  is  now 
well  known.  It  is  evident  that  they  are  reserve  substances.  Their 
evolution  proves  it.  Thus  metachromatic  corpuscles  appear  in  great 
abundance  in  the  young  asci  of  the  higher  Ascomycetes  (Fig.  29,  i  and  2), 
then  accumulate  in  the  cytoplasm  of  the  epiplasm  which  is  not 
utilized  in  the  formation  of  the  ascospores,  gather  all  around  the 
ascospores  at  the  time  of  their  forming  (3-4),  and  are  gradually 


../•••*•       <"2 
FIG.  30. — Conidial  organ  of  Aspergillus  niger  with  metachromatic  corpuscles. 

absorbed  by  the  latter  in  the  course  of  their  development  (5). 
They  therefore  furnish  nourishment  for  the  ascospores  and  from  this 
standpoint  behave  exactly  like  glycogen  and  the  globules  of  fat 
which  are  usually  coexistent  with  them  in  the  cytoplasm.  We 
shall  see,  moreover,  that  they  undergo  a  similar  evolution  in  the 
asci  of  yeasts.  Likewise  in  the  conidiophores  of  molds,  notably  in 
the  fruiting  heads  of  Aspergillus  and  Penicillium,  the  metachromatic 
corpuscles  are  produced  in  great  abundance  (Figs.  30  and  31),  then 
gradually  disappear  as  the  conidia  form  (30,  3).  Here  again  they 
serve  as  food  for  the  conidia. 

1Because  of  the  priority  and  more  exact  signification,  the  names  metachromatic  corpuscles 
and  metachromatin  are  preferable  to  the  terras  grains  of  volutin  and  volutin  given  by  Arthur 
Meyer. 


46  MORPHOLOGY  AND    CULTURE    OF  MICROORGANISMS 

Metachromatic  corpuscles  appear  not  only  in  the  vacuoles,  but  also 
in  the  perivacuolar  cytoplasm.  There  they  spring  up,  to  diffuse  finally 
in  the  vacuole  where  they  increase.  It  is  difficult  to  observe  their 
manner  of  forming  in  the  mycelial  filaments,  but  in  the  preparation  for 
sporulation  in  some  molds  (asci  of  the  higher  Ascomycetes),  it  has 
recently  been  demonstrated  that  they  start  in  the  midst  of  the  elements 
of  the  chondrium,  which  act  as  plastids  similar  to  the  plastids  of  the 
higher  plants.  They  start  in  the  interior  of  the  granular-mitochondria 
or  in  the  rod-mitochondria  (Fig.  31).  In  the  former  case,  a  small  cor- 


o> 

XT 

FIG.  31.  —  Formation  of  metachromatic  corpuscles  in  a  cell  of  the  perithecium  of 
Pestularia  vesiculosa.  The  rod-mitochondria  form,  on  their  crossings,  vesicles  (c) 
consisting  of  a  metachromatic  corpuscle  unstained  by  the  special  method  which 
served  to  differentiate  the  chondrium.  Some  corpuscles  (a),  more  highly  developed, 
are  found  in  the  vacuoles  still  surrounded  by  their  mitochondrial  shell;  others  (c) 
at  the  completion  of  their  development  have  worn  through  their  mitochondrial 
covering. 

puscle  appears  in  the  substance  of  a  mitochondrium,  then  develops 
gradually,  while  the  mitochondrial  membrane  which  envelops  it  grows 
thinner;  is  reduced  to  a  small  capping  of  the  grain  on  one  side;  then 
disappears  when  the  latter  reaches  maturity.  It  is  noteworthy  that  the 
corpuscles  emigrate  with  their  plastid  to  the  interior  of  the  vacuoles 
during  their  development. 

When  the  corpuscles  start  in  a  rod-mitochondrium  the  process  is 
much  the  same  as  in  the  granular-mitochondria;  when  several  rod- 
mitochondria  are  involved,  at  their  junction  small  corpuscles  are 
seen  to  form;  the  parts  of  the  rod-mitochondria  which  join  are  then 
absorbed  and  the  corpuscles,  enclosed  in  their  mitochondrial  membrane, 
once  separated,  undergo  the  same  evolution  as  above. 

Thus  the  metachromatic  corpuscles,  like  grains  of  starch  in  the 
higher  plants,  start  in  the  midst  of  the  mitochondria  and  develop 
gradually  out  of  their  mitochondrial  matrix  with  the  aid  of  the  vacuolar 
substance, 


MOLDS  47 

In  molds  are  found  still  other  reserve  products.  One  often  sees 
globules  of  fat  in  the  cytoplasm,  which  are  easily  stained  by  a  black- 
brown  by  osmic  acid;  and  glycogen  which  can  be  differentiated  by  iodine 
in  iodide  of  potassium.  The  glycogen  is  contained  in  either  the 
cytoplasm  or  the  vacuoles.  It  is  generally  very  abundant. 

These  products  (fat  and  glycogen)  undergo  the  same  evolution  as 
the  metachromatic  corpuscles,  and  they  also  accumulate  in  the  organs 
of  fructification  (asci,  conidial  organs)  to  serve  in  the  nourishment  of 
spores  and  conidia. 

CELL- WALL. — The  cell-wall  of  molds  is  quite  distinct  and  often 
thick.  It  is  sometimes  cutinized.  According  to  Mangin,  it  consists 
of  callose  and  pectose  with  which  is  often  associated  a  kind  of  cellulose. 

SPECIFIC  CONSIDERATION  OF  MOLDS* 

A  few  species  are  found  to  grow  very  constantly  in  the  same  situa-. 
tions  as  bacteria.  These  are  associated  with  forms  of  decay,  fermenta- 
tion, or  disease,  either  as  primary  or  secondary  causes.  They  thus 
become  important  to  the  bacteriologist  who-  studies  them  by  the  same 
methods  as  bacteria.  These  species  belong  to  widely  scattered  groups 
of  fungi,  so  that  species  found  under  the  same  conditions  frequently 
differ  greatly  in  appearance.  The  common  term,  molds,  is  applied 
collectively  to  these  organisms,  though  no  sharp  limits  can  be  set  to 
the  use  of  the  term.  Physiologically  these  species  can  be  considered 
in  three  series: 

COSMOPOLITAN  SAPROPHYTES. — Certain  species  are  capable  of 
growing  within  very  wide  limits  of  temperature  and  of  composition  of 
substrata.  Many  of  these  have  accompanied  man  everywhere  and  are 
constantly  found  upon  every  kind  of  putrescible  matter,  especially  as 
the  causes  of  fermentation  or  decay  in  food.  Their  spores  (conidia)  are 
produced  in  countless  numbers,  and  are  so  light  that  they  float  hi  air 
currents  and  are  carried  by  contact  in  every  conceivable  manner  by 
animals  and  by  man.  The  life  cycle  from  spore  to  spore  is  frequently 
very  short,  often  being  completed  in  twenty-four  hours  or  less.  Many 
of  these  forms  are  propagated  for  an  indefinite  number  of  generations  by 
asexual  spores  or  conidia  but  produce  sexual  fruit  when  special  conditions 
are  furnished.  Some  of  them  have  never  been  induced  to  develop  a 

*  Prepared  by  Charles  Thorn. 


48  MORPHOLOGY   AND    CULTURE   OF   MICROORGANISMS 

"perfect"  form.  These  species  are  the  " weeds"  of  the  bacterial 
culture-room,  since  they  cannot  be  entirely  eliminated  and  often  times 
will  survive  conditions  more  severe  than  the  bacteria  themselves. 

MOLDS  OF  FERMENTATION. — A  few  species  have  acquired  special 
importance  by  their  fermentative  action.  Certain  of  these  forms  are 
widely  distributed  and  able  to  utilize  other  media  and  conditions. 
They  differ  from  closely  related  species  of  the  same  genera  in  the  ability 
to  produce  special  enzymes  or  especially  large  amounts  of  such  enzymes 
as  bring  about  particular  forms  of  fermentation.  Certain  of  these 
species  have  been  utilized  in  the  manufacture  of  drinks,  of  citric  acid, 
in  cheese  ripening,  etc.  Others  are  so  adapted  to  growth  under  condi- 
tions of  fermentation  as  to  be  found  constantly  in  connection  with  such 
processes,  in  which  their  vigorous  growth  and  fermenting  power 
seriously  interferes  with  control  of  results. 

PARASITIC  MOLDS.- — Many  species  of  molds  have  been  described 
as  the  cause  of  diseases  in  man  and  domestic  animals.  A  few  of 
these  forms  have  been  isolated  and  studied.  Some  of  them  attack 
the  lungs,  others  the  kidneys,  but  the  larger  number  appear  as  the 
cause  of  diseased  areas  (dermatomycoses)  of  the  skin,  hair  follicles, 
and  external  ears,  or  swellings  or  malformations  of  the  extremities. 
Of  a  large  number  of  forms  named  from  a  microscopic  determina- 
tion of  their  presence  in  particular  lesions,  only  a  few  have  been 
adequately  characterized  and  shown  to  be  primary  agents  in  caus- 
ing the  injuries  observed.  Aspergillus  fumigatus,  various  species  of 
Sporotrichum  and  Actinomyces,  the  scalp  organisms  of  herpes  and 
favus  appear  to  be  real  pathogens. 

GENERIC  CONSIDERATION  OF  GROUPS* 

THE  MUCORS  OR  BLACK  MOLDS. — The  mucors  or  black  molds  con- 
stitute a  large  group  of  species  belonging  to  the  Phycomycetes  or  algal 
fungi  whose  general  characters  are  a  unicellular  mycelium,  at  least  in  the 
vegetative  stage,  and  quite  generally  a  well-developed  form  of  sexual 

*The  series  of  forms  presented  contains  representatives  of  the  most  common  groups  as 
they  occur  in  laboratory  cultures,  and  such  as  have  acquired  importance  to  the  worker  in 
bacteriology  by  participation  in  processes  regularly  studied  by  the  bacteriologist.  For  more 
complete  discussion  of  the  fungi,  the  student  is  referred  to  standard  text-books  of  cryptogamic 
botany.  For  discussions  of  species,  Lafar's  Technical  Mycology  includes  the  groups  found 
associated  with  the  bacteria;  for  other  groups,  special  botanical  literature  must  be  consulted. 


MOLDS 


49 


reproduction  (Figs.  32  and  33).  In  the  mucors,  the  mycelium  is  usually 
richly  developed  within  and  often  also  on  the  surface  of  the  substratum; 
asexual  reproduction  is  accomplished  by  spores  borne  as  conidia  or 
borne  within  sporangia;  and  sexual  reproduction  is  accomplished  by 
the  conjugation  of  special  branches  from  the  mycelium  forming  zygo- 


FIG.  32.  FIG.  33. 

FIG.  32. — Mucorineos.  Mucor.  From  Tabula  Botanical,  showing  sporangia 
originating  from  mycelium,  spores  and  spore  germination,  and  the  formation  of 
zygospores  in  a  heterothallic  species  (diagrammatic).  (Reduced  one-half.)  (By 
permission  of  A.  F.  Blaskeslee.) 

FIG.  33. — MuLorinea.  Mucor,  Rhizopus.  A,  B,  C,  D,  Formation  of  the  zygo- 
spores from  conjugating  branches;  E,  section  of  Z>;  F,  mature  zygospores  in  section; 
G,  germination  of  zygospores;  H,  diagram  pf  fruiting  stolons  of  Rhizopus  nigricans; 
K,  section  of  sporangium  during  spore  formation,  highly  magnified  (From  Tabula 
Botanica.)  (Reduced  one-half.)  (By  permission  ofA.F.  Blaskeslee.} 

spores  (Figs.  32  and  33).  The  typical  mucors  produce  sporangia  as 
capsule-like  dilations  at  the  ends  of  erect  fertile  hyphae,  each  con- 
taining many  spores.  Septa  are  commonly  developed  in  the  mycelium 
when  sporangia  begin  to  appear.  These  fertile  hyphae  may  be  micro- 
scopic or  attain  a  length  of  several  centimeters. 

Important  Species. — Perhaps    the    commonest   form   is   Rhizopus 
nigricans  (syn.  Mucor  stolonifer),  the  black  mold  of  bread,  a  cosmo- 

4 


50  MORPHOLOGY  AND   CULTURE   OF   MICROORGANISMS 

politan  species  associated  with  the  decay  of  many  kinds  of  food  stored 
in  wet  condition  or  in  humid  situations.  Typical  clusters  of  spor- 
angiophores  are  borne  on  stolons  or  runners,  which  are  hyphae  extending 
radially  from  the  center  of  the  colony  and  fastened  to  the  substratum 
or  to  the  support  at  intervals  by  root-like  outgrowths.  Abundant 
growth  of  this  species  is  found  only  under  very  moist  conditions  or 
in  substrata  with  high  water  content.  Rhizopus  is  a  very  common 
contamination  in  laboratory  cultures. 

Many  species  and  races  of  Rhizopus  have  been  described.  These 
have  been  studied  especially  in  connection  with  the  fermentation 
industries  of  Japan  and  China.  Rice,  wheat,  and  soy-beans  in  various 
mixtures  pass  through  an  initial  process  of  "Koji"  preparation  in  which 
raw  or  cooked  materials  are  exposed  to  the  air  for  several  days.  These 
processes  offer  ideal  conditions  for  the  entrance  of  the  mucors  as 
contaminations  among  the  organisms  desired. 

There  are  many  common  species  of  the  genus  Mucor,  very  few  of 
which  are  identifiable  without  critical  study.  The  specific  names  as 
commonly  cited  often  designate  groups  of  species  or  varieties  rather 
than  sharply  marked  forms.  Certain  of  these  may  be  briefly  considered. 

Mucor  mucedo  L.  -is  a  common  form  upon  dung,  characterized  by 
heads  (sporangia)  upon  long  sporangiophores,*  at  first  yellow  then 
becoming  dark  brown  or  black  and  studded  upon  the  surface  with 
needles  of  lime. 

Mucor  racemosus,  Fresenius,  is  characterized  by  the  production  of 
chlamydospores  or  cysts  in  the  mycelium  within  the  substratum,  as 
elliptical  thick-walled  cells.  The  sporangiophores  typically  branch  to 
make  racemes  of  sporangia.  The  racemose  mucors  are  active  agents  in 
changing  starch  to  sugar  and  in  the  production  of  traces  at  least  of 
alcohol  from  sugars. 

Mucor  rouxii  (Calm.),  Wehmer,  (syn.  Amylomyces  rouxii}  is  the 
most  important  of  a  series  of  forms  with  sporangiophores  branching 
sympodially  which  are  active  in  changing  starch  to  sugar  and  in  produc- 
ing traces  at  least  of  alcohol.  The  mycelium  of  Mucor  rouxii  develops 
in  fluid  cultures  as  yeast-like  cells  and  groups  of  cells.  The  typical 

*The  term  sporangiophore  is  composed  of  the  word  sporangium  combined  with  the  suffix 
phore,  meaning  bearer.  In  sympodial  branching  the  first  fruit  is  on  the  tip  of  the  original  hypha, 
the  first  branch  arises  below  this  fruit  and  is  terminated  by  the  second  fruit.  Each  successive 
branch  and  fruit  originates  in  similar  manner. 


MOLDS  51 

mucor  fruits   are  produced  only  under  special   cultural  conditions. 
This  organism  is  used  in  the  amylo  process  of  alcoholic  fermentation. 

Fermentation  activity  has  been  described  for  numerous  species  of 
Mucor  and  Rhizopus.  Among  them  are  Mucor  circinelloides,  Van 
Tieghem,  Mucor  javanicus,  Wehmer,  Mucor  plumbeus,  Bonorden, 
Rhizopus  oryzce,  Went,  Rhizopus  javanicus.  The  fermenting  power 
of  mucors  like  that  of  yeasts  varies  greatly  with  the  species  or  even 
with  races  used,  approaching  in  some  species  the  efficiency  of  the 
more  active  yeasts. 

THAMNIDIUM. — Of  related  genera,  Thamnidium  differs  from  Mucor 
in  the  production  of  two  kinds  of  sporangia.  The  terminal  sporangium 
of  a  fruiting  hypha  resembles  that  of  Mucor;  the  secondary  or  accessory 
sporangia  which  are  borne  upon  side  branches  of  the  sporangiophores 
are  smaller,  lack  the  columella,  and  produce  few  to  several  spores 
within  an  outer  wall. 

Thamnidium  elegans,  Link,  produces  primary  and  secondary  spor- 
angia on  different  hyphae,  together  making  white  colonies.  The  fertile 
side  branches  are  produced  in  whorls  and  bear  whorls  of  branchlets 
from  their  centers  which  in  turn  produce  sporangioles  from  the  tips  of 
short  straight  twigs  or  branchlets. 

PENICILLIUM. — The  extremely  abundant  green  molds  .  most  fre- 
quently belong  to  the  genus  Penicillium,  although  some  members  of 
other  groups  may  be  confused  with  them  at  times. 

Characters. — Colonies  are  composed  of  loosely  woven  hyphse, 
branched,  septate,  colorless,  or  bright  colored.  The  fertile  hyphae 
(conidiophores)  are  mostly  erect,  arising  either  from  submerged  hyphae, 
or  as  branches  of  aerial  hyphae,  septate,  usually  branched  only  in  the 
fruiting  portion.  Conidial  fructifications  consist  of  more  or  less  com- 
plex systems  of  branches  and  branchlets,  the  ultimate  fertile  cells  each 
producing  a  chain  of  conidia  (Fig.  34).  The  whole  system  is  usually 
grouped  near  the  end  of  the  conidiophore,  giving  the  appearance  of 
one  or  more  brooms  or  brushes  (whence  the  name).  Very  few  species 
are  known  to  produce  asci,  hence  these  are  rarely  encountered.  The 
conidial  form  continues  for  an  indefinite  number  of  generations,  there- 
fore all  the  activities  of  the  genus  are  associated  with  this  form.  The 
classification  of  the  whole  group,  technically  transferred  by  some 
workers  to  the  ascomycetes  on  account  of  certain  forms,  becomes 
misleading,  because  it  contains  so  few  species  producing  asci. 


52  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

So  far  as  evidence  has  accumulated  nearly  all  the  forms  met  are 
imperfect. 


FIG.  34. — Penicillium  expansum,  Link,  a,  b,  /,  Branching  and  arrangement  of 
branches  of  conidial  fructification  (Xgoo);  c,  d,  e,  conidiiferous  cells  and  conidial 
chains  (Xgoo);  g,  h,  j,  k,  I,  sketches  of  fructifications  (Xi4o);  m,  n,  o,  germination 
of  conidia  (Xgoo);  r,  s,  sketches  from  photographs  showing  in  s  loose  aggregations 
of  conidiphores  beginning  to  develop  into  zonately  arranged  coremia,  in  r  a 
coremium  i  mm.  in  height.  (From  Bui.  118,  Bureau  of  Animal  Industry,  U.  S. 
Dept.  Agriculture.} 


Cultural  Considerations. — Among  the  numerous  species  and  races 
are  certain  green  forms  which  are  widely  distributed  and  almost  om- 
nivorous in  habit.  Other  species  are  closely  restricted  to  particular 


MOLDS  53 

substrata.  Starches  and  sugars  appear  to  be  especially  favorable 
components  of  nutrient  media  for  members  of  the  group.  The  larger 
number  of  the  species  grows  best  at  temperatures. from  15°  to  30°; 
a  very  few  of  them  reach  their  optimum  at  37°,  but  many  species  are 
entirely  inhibited  and  some  killed  at  blood-heat.  Vegetative  mycelium 
begins  to  be  produced  at  temperatures  very  close  to  freezing,  but 
colored  conidia  are  produced  slowly  or  not  at  all  at  low  temperatures. 
The  species  of  Penicillium  thrive  through  a  wide  range  of  concentration 
of  culture  media,  though  perhaps  the  most  characteristic  growths  are 
produced  in  media  high  in  water  content.  The  common  species  of 
this  genus  grow  in  all  the  standard  bacteriological  media.  With  few 
exceptions  .  the  species  grow  well  in  synthetic  media  composed  of 
assimilable  carbohydrates  and  inorganic  salts.  A  few  species  require 
the  presence  of  some  one  of  the  higher  nitrogenous  compounds,  but 
many  species  refuse  to  produce  typically  colored  fruit  without  some 
form  of  starch  or  sugar  in  addition  to  ordinary  peptone  and  beef- 
extract.  Very  few  species  grow  well  in  alkaline  media,  but  most 
species  are  tolerant  of  organic  acids  at  the  concentrations  found  in 
fruits  and  vegetables. 

Some  Common  Species. — Penicillium  roqueforti,  Thorn,  is  a  green 
form  constantly  found  in  pure  culture  in  Roquefort  cheese,  frequently 
also  in  ensilage.  It  is  widely  distributed  and  grows  under  many  sets 
of  conditions. 

Penicillium  camemberti,  Thorn,  is  the  chief  organic  agent  in  ripening 
Camembert  cheese.  Cultures  of  this  species  are  floccose  or  cottony, 
at  first  white,  later  gray-green. 

Penicillium  expansum,  Link,  is  a  green  form,  always  obtainable  from 
apples  decaying  in  storage,  upon  which  it  frequently  produces  large 
coremia  or  stalks  bearing  conidia  in  masses  sometimes  several  millimeters 
in  diameter.  It  is  one  of  the  most  abundant  species  of  the  genus, 
widely  distributed  in  different  countries.  In  cultures,  colonies  produce 
a  characteristic  odor,  suggestive  of  its  common  habitat,  decaying  apples. 

Penicillium  brevicaule,  Saccardo,  (Scopulariopsis  repens,  Bainier) 
is  a  form  with  rough  or  spiny  brown  spores  which  has  been  used  phy- 
siologically to  detect  the  presence  of  arsenic  by  its  ability  to  set  free 
arsine  from  such  substrata.  A  whole  series  of  forms  has  since  been 
found  to  possess  this  character  correlated  with  characteristic  spore 
formation.  These  species  or  races  are  common  in  the  soil  both  in 


54  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

Europe  and  America  and  appear  as  frequent  contaminations  upon 
cheese  and  upon  cured  meats  to  both  of  which  products  they  impart 
peculiarly  penetrajting  ammoniacal  flavors.  One  member  of  this  group 
has  been  reported  as  present  in  war- wounds. 

Except  species  associated  with  particular  processes  or  substrata, 
the  identification  of  the  green  species  of  Penicillium  requires  special 
methods  and  greater  care  than  is  possible  aside  from  special  study  of 
the  group. 

ASPERGILLUS  (AND  STERIGMATOCYSTIS). — The  genus  Aspergillus  in- 
cludes numerous  species  which  develop  under  widely  different  condi- 
tions. Many  of  these  forms  reach  their  typical  development  under 
drier  conditions  than  Penicillium  and  Mucor,  such  as  stored,  grain,  her- 
barium specimens,  dried  flesh,  or  foods  containing  concentrated  sugars, 
such  as  jams,  jellies,  etc.  Some  excite  processes  of  fermentation,  and 
a  few  are  associated  with  diseases. 

Characters. — The  vegetative  hyphae  are  creeping,  submerged  in  the 
substratum  or  sometimes  aerial  also,  branched,  septate,  usually  color- 
less, and  sometimes  bright  colored.  Conidiophores  or  fertile  hyphae 
arise  by  transformation  of  single  hyphal  cells  into  thick-walled  and 
often  characteristically  shaped  foot-cells  from  which  the  fertile  stalks 
arise  as  perpendicular  branches  which  are  erect,  unseptate,  or  few- 
septate,  usually  much  larger  in  diameter  than  the  vegetative  hyphae, 
and  gradually  enlarged  upward,  ending  in  more  or  less  abrupt  dilations 
or  heads  which  bear  closely  packed  columnar  sterigmata  or  conidiif  erous 
cells  over  the  whole  or  a  large  part  of  their  surface  (Fig.  35,  ft).  Each  of 
these  cells  bears,  in  one  group  of  species,  a  single  chain  of  conidia;  in 
other  species  (called  by  some  authorities  Sterigmatocystis)  all  or  part  of 
these  sterigmata  bear  several  secondary  sterigmata  which  bear  the 
conidial  chains.  Part  of  the  species  produce  also  thin-walled  perithecia 
as  variously  colored  spherical  bodies  upon  the  surface  of  the  substrata. 
These  perithecia  are  filled  with  eight-spored  asci  (Fig.  35,  e).  Species 
in  certain  groups  produce  sclerotia  instead  of  perithecia,  but  many 
species  are  not  known  to  produce  either  perithecia  or  sclerotia. 

Important  Species. — Among  the  species  constantly  met  with, 
A  spergillus  niger  is  recognizable  by  its  black  or  very  dark  brown  spores 
and  in  some  strains  by  black  sclerotia.  Several  black-spored  forms  are 
described,  but  their  separation  is  usually  impossible  by  ordinary 
methods  of  culture.  Aspergillus  niger  ferments  sugar  solutions  with 


MOLDS 


55 


the  production  of  oxalic  acid  in  considerable  quantity.  Citric  acid 
fermentation  with  Aspergillus  niger  has  been  successfully  developed 
upon  a  factory  basis  by  Currie  in  the  United  States. 

Of  green  forms,  Aspergillus*  glaucus,  Link  (Aspergillus  herbariorum, 
Wiggers),  and  Aspergillus  repens,  De  Bary,  both  produce  abundant 
yellow  perithecia.  These  abound  upon  herbarium  specimens,  hay, 
grain,  concentrated  foods,  such  as  jellies,  preserves,  dried  bread  and 
dried  meats  upon  which  they  produce  green  conidial  areas  which  are 
later  dotted  with  bright  yellow  perithecia. 


FIG.  35.  FIG.  36. 

FIG.  35. — Aspergillus  glaucus.  a,  Conidiophore  showing  increased  diameter 
over  the  vegetative  cells  at  its  base  (Xi28);  b,  sterigmata  (X45o);  c,  conidia,  smooth 
thick  walled  in  this  variety,  other  varieties  are  spiny  (X45o);  d,  perithecium(Xi28); 
e,  ascus  containing  ascospores  (X45°)-  (Original.) 

FIG.  36.— Aspergillus.  (i)  A.  fumigatus,  Fres;  (2)  A.  nidulans.  i  and  2,  Show 
the  simple  sterigmata  of  A.  fumigatus  and  the  secondary  sterigmata  of  A.  nidulans. 
The  conidia  of  these  species  do  not  remain  attached  in  ordinary  fluid  mounts. 
(Original.) 

Aspergillus  fumigatus,  Fresenius,  is  a  green  form  characterized  by 
short  conidiophores  enlarging  gradually  into  heads  and  bearing  a  single 
set  of  sterigmata  on  the  very  apex,  with  chains  of  thin-walled  green 
spores  about  3A*t  m  diameter.  This  species  produces  a  destructive 
disease  of  birds  known  as  aspergillosis.  The  same  species  is  sometimes 
reported  as  pathogenic  to  man. 

Aspergillus  nidulans  differs  by  having  two  sets  of  sterigmata  but 
otherwise  frequently  closely  resembles  Aspergillus  fumigatus.  It  is 

*Recent  examination  of  a  large  number  of  American  specimens  shows  that  Aspergillus 
repens  is  the  usual  green  form  in  this  country. 

fThe  unit  of  measurement  is  the  micron  GO  or  micro-millimeter  (.001  mm.  or  Hiooo  in.) 


56  MORPHOLOGY  AND    CULTURE    OF   MICROORGANISMS 

widely  distributed  in  soil.  Although  it  has  been  reported  as  pathogenic 
the  identification  of  this  species  in  pathogenic  lesions  is  not  confirmed. 

Aspergillus  oryzce  and  A.  flaws  form  a  closely  intergrading  series, 
certain  members  of  which  are  used  in  the  fermentation  industries  of 
Japan  and  China.  A.  oryzce  as  described  by  Wehmer  is  used  in  the 
fermentation  of  rice  to  produce  Sake,  an  alcoholic  drink.  The  diastatic 
enzyme  of  A.  oryzce  grown  upon  rice  converts  the  starch  into  sugars 
which  are  fermented  by  yeasts  into  alcohol.  Other  members  of  the 
series  more  closely  approximating  A.  flavus  are  widely  used  in  the 
fermentation  of  soy-beans.  A  mixture  of  cooked  soy-beans  and 
cracked  roasted  wheat  is  inoculated  with  A.  flavus  in  special  koji 
fermenting  chambers.  In  three  days  the  entire  mass  becomes  fully 
overgrown  with  mycelium  and  covered  with  the  ripe  conidia  of  this 
form.  The  wheat  and  beans  are  partially  penetrated  by  the  mold 
hyphae.  The  mass  is  then  transferred  to  brine  strong  enough  to  inhibit 
further  growth  except  of  a  few  yeasts.  In  a  long  period,  several 
months  to  three  years,  the  whole  mass  becomes  digested  to  form 
soy-sauce,  or  shoyu.  This  product  is  the  basis  of  meat  sauces  such  as 
Worcestershire. 

Aspergillus  wentii,  Wehmer,  characterized  by  its  long  conidiophores 
and  coffee-colored  heads  of  conidia,  is  found  in  the  Soja  preparation  in 
Java. 

Of  other  forms  constantly  met,  Aspergillus  candidus  has  white  or 
pale  cream  fruiting  surfaces.  A.  terreus  is  avellaneous  in  color;  Asper- 
gillus ochraceus,  ocher  or  tan. 

Much  confusion  is  still  found  in  the  literature  of  this  genus,  so  that 
frequent  references  to  the  activities  of  particular  species  are  difficult  or 
impossible  to  verify. 

MONASCUS. — The  organism  of  red  ensilage  is  widely  distributed  in  the 
silos  of  America.  Ensilage  infected  with  Monascus  forms  into  red  balls 
or  masses  up  to  a  foot  in  diameter  held  together  by  mycelium.  The 
masses  are  red  from  coloring  material  partly  in  the  mycelium  and  spore- 
masses  and  partly  in  the  silage.  The  same  or  a  nearly  related  form 
described  as  Monascus  purpureus,  Went,  is  used  in  producing  red  rice, 
A  ng-quac,  in  China.  The  mycelium  penetrates  the  Vice  grains,  produces 
a  friable  texture  and  gives  the  whole  mass  a  purple  red  color.  Ang- 
quac  (or  Ang-khak)  is  used  to  color  Chinese  sauces  and  reaches  America 
especially  upon  Chinese  soy-bean  cheeses. 


MOLDS 


57 


CLADOSPORIUM  (AND  HORMODENDRON). — The  species  of  Cladospor- 
ium  occur  frequently  in  cultures  of  decaying  vegetable  matter,  of  milk 
and  cream,  or  butter.  The  colonies  liquefy  gelatin.  Both  mycelium 
and  spores  are  at  first  colorless,  but  later  dark  colored  to  almost  black, 
with  spores  becoming  two-celled  in  very  old  cultures. 

-  Cladosporium  herbarum  is  the  commonest  species  encountered.* 
Colonies  in  culture  media  differ  so  greatly  in  structure  from  those  upon 
natural  substrata  as  to  make  identification  of  species  questionable. 
(Fig.  37).  Much  confusion  is  therefore  found  in  the  use  of  the  names 
of  species  of  Cladosporium  and  the  related  genus,  Hormodendron,  which 
is  separated  by  some. 


FIG.  37.  FIG.  38.  FIG.  39. 

FIG.  37. — Cladosporium  herbarum,  showing  the  forms  of  conidiophores  and  conidia 
which  are  very  common  upon  laboratory  culture  media.  (Original.) 

FIG.  38. — Spores  of  Alternaria  sp.     (Original.) 

FIG.  39. — Fusarium  from  decaying  potato,  a,  Spores  showing  curvature  and 
septa;  b,  germination  of  spores;  c,  development  of  spores  in  petri-dish  culture;  d, 
mass  of  spores  as  found  in  culture.  (Original.) 

ALTERNARIA  AND  FUSARIUM. — The  frequent  occurrence  of  species  of 
Alternaria  and  Fusarium  in  cultures  demands  that  the  generic  characters 
be  recognized.  Both,  as  a  rule,  produce  abundant  growth  with  a  tend- 
ency to  over-run  cultures  of  other  forms  (Figs.  38,  39).  The  spores  of 
Alternaria  are  brown,  Indian-club  form  or  muriform  (divided  into 
several  cells  by  longitudinal  as  well  as  cross  walls),  and  are  connected 
together  into  chains  (Fig.  38).  The  spores  of  Fusarium  are  colorless, 
either  straight,  sickle-shaped,  or  crescent-shaped,  divided  into  several 
cells  by  cross  walls,  are  produced  in  chains  or  adhere  into  masses  on  the 
tips  of  the  fertile  branchlets.  The  morphology  of  colonies  in  culture 
varies  widely  from  the  descriptions  of  the  same  species  under  natural 
conditions.  Species  of  Fusarium  frequently  produce  bright  colors  in 

*  This  species  has  been  shown  to  be  a  conidial  form  of  Spaerella  tulasnei  Janczewski,  but  the 
bacteriological  student  will  meet  only  the  conidial  stage. 


58  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

the  mycelium  and  substrata;  colonies  of  Alternaria  often  become  almost 
black.  Identification  of  species  in  cultures  is  thus  far  impossible, 
except  for  the  specialist. 

OIDIUM. — Oidium  (Oospora)  lactis  is  universally  found  in  cultures 
from  milk  and  milk-products  and  occurs  very  frequently  in  decaying 
vegetables,  manure,  etc.  Colonies  of  the  species  are  colorless,  have 


FIG.  40.  —  Oidium  lactis.  a,  b,  Dichotomous  branching  of  growing  hyphae;  c,  d, 
g,  simple  chains  of  oidia  breaking  through  substratum  at  dotted  line  x-y,  dotted 
portions  submerged;  e,f,  chains  of  oidia  from  a  branching  out-growth  of  a  submerged 
cell;  h,  branching  chain  of  oidia;  k,  I,  m,  n,  o,  p,  s,  types  of  germination  of  oidia  under 
varying  conditions;  t,  diagram  of  a  portion  of  a  colony  showing  habit  of  Oidium 
lactis  as  seen  in  culture  media.  (From  Bull.  82,  Bur.  Animal  Industry,  U.  S.  Dept. 


vegetative  mycelium  entirely  submerged,  become  powdery-white  with 
spores  when  mature,  liquefy  gelatin,  and  produce  a  strong  character- 
istic odor  (Fig.  40).  Microscopically  the  species  is  recognized  by  dicho- 
tomous  branching  of  the  hyphae  at  the  margin  of  the  rapidly  growing 
colonies,  and  by  the  spores  or  oidia  which  are  abruptly  cylindrical, 
varying  with  conditions  in  length  and  diameter  and  produced  both 


MOLDS  59 

above  and  below  the  surface  of  the  substratum  in  long  chains  which 
break  up  readily.  At  times  the  whole  mycelium  appears  to  break  up 
into  oidia.  Oidium  lactis  is  a  factor  in  the  ripening  of  many  kinds  of 
cheese:  Limburger,  Harz,  Camembert,  Gorgonzola,  etc.,  and  in  the 
deterioration  of  butter  in  storage.  Its  activity  is  associated  with 
strong  odor  and  taste. 


FIG.  41. — A  colony  of  Monilia  Candida.     (Photographed  by  Z.  Northrup.) 


FIG.  42. — Forms  of  oidia  in  chains.     (Monilia  sitophila.} 

MONILIA. — The  generic  name  Monilia  is  very  loosely  used  in  the 
fermentation  literature  for  certain  forms  in  which  the  conspicuous 
development  consists  of  chains  of  cells  like  strings  of  beads.  There  is  a 
series  of  borderline  forms  between  the  true  yeasts,  the  mycoderma 
group  of  yeasts  and  the  true  hyphal  fungi.  The  name  Monilia  Candida 


60  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

was  applied  by  Hansen  to  one  of  these  which  is  found  frequently  in 
breweries.  The  genus  Willia  has  been  created  for  another  series  of 
these  forms.  Another  widely  distributed  species,  Monilia  sitophila,  forms 
loose  salmon-pink  masses  of  conidia  on  .the  surface  and  in  the  interior  of 
bread,  in  cereals  and  other  foods.  In  culture  media  Monilia  sitophila 
fills  culture  tubes  and  dishes  with  loose  fluffy  salmon  masses  of  conidia. 
This  organism  frequently  overruns  an  incubator  or  a  culture  room  in- 
fecting everything  fermentable. 

DEMATIUM. — One  species  of  Dematium,  Dematium  pullulans,  has 
been  much  studied.  This  is  frequently  found  within  decaying  fruit  as 
dark  brown  colonies.  In  culture,  mycelium  is  sparingly  produced, 
either  colorless  or  colored,  and  conidia  are  borne  in  clusters  and  chains 
all  along  the  hyphae  submerged  in  the  substratum.  At  first  both  myce- 
lium and  conidia  are  colorless,  later  some  or  all  of  the  cells  develop 
heavy  dark  brown  walls.  Although  not  active  as  an  agent  of  fermen- 
tation, it  occurs  very  frequently  in  the  fermentation  industries  some- 
times discoloring  the  fermenting  products.  The  conidia  bud  out  from 
the  cells  of  the  mycelium  in  a  manner  resembling  the  yeasts.  Its 
occurrence  with  the  yeasts  has  led  to  many  careful  descriptions  of  its 
several  types  of  spore  production  and  its  biological  activities. 

SAPROLEGNIACE/E. — This  is  an  aquatic  group  of  Phy corny cetes,  which 
includes  both  saprophytes  and  parasites.  Its  commonest  members 
grow  as  shimmering  masses  of  cottony  mycelium  upon  the  bodies  of 
flies  or  other  insects  in  aquaria.  Other  members  of  the  same  group 
are  parasitic,  some  attacking  young  fish  and  producing  characteristic 
lesions.  Both  sexual  and  asexual  spores  (motile  swarm  spores)  are 
abundantly  found. 


CHAPTER  III 

YEASTS* 

MORPHOLOGY  OF  CERTAIN  TYPES 

DEFINITION  AND  BASES  OF  CLASSIFICATION. — If  the  cloudy  freshly 
expressed  juice  of  grapes  or  other  fruits  be  passed  through  a  centrifuge, 
the  sediment  will  be  found  to  consist  principally  of  amorphous  particles 
of  dirt  and  plant  tissue.  If  the  clear  juice  is  now  allowed  to  stand  in  a 
warm  place  for  a  few  days  it  will  ferment  and  the  sediment  thrown 
down  by  the  centrifuge  may  be  shown  by  the  microscope  to  consist  prin- 
cipally of  unicellular  microorganisms. 

These  microscopic  cells  are  called  collectively  "yeast"  and  belong 
to  various  groups  of  fungi.  Some  of  them  are  special  vegetative  forms 
of  Phy  corny  cetes  (Mucor),  others  of  Ascomycetes  (Saccharomyces,  Asper- 
gillus),  while  others  are  unknown  in  any  other  form  and  are  classed  as 
Fungi  imperfecti  (My  coder  ma,  Torula).  They  are  widely  distributed  in 
nature  and  some  of  them  occur  on  all  exposed  surfaces  and  particularly 
on  moist  organic  substances  containing  sugar  and  acid.  The  true 
yeasts  (Saccharomy cetes),  which  are  of  the  greatest  importance  indus- 
trially, occur  naturally  on  the  raw  material  (S.  ellipsoideus  on  grapes) 
or  are  known  best  in  the  cultivated  condition  (S.  cerevisia  of  beer). 

The  true  yeasts  occur  in  the  form  of  spherical  or  more  or  less  elon- 
gated cells  varying  in  normal  width  from  2.5/1  to  i2/i.  The  first  classi- 
fications were  based  on  shape  and  size  alone  but  these  vary  and  depend 
so  much  on  cultural  conditions  that  they  are  of  little  value  in  differen- 
tiating species  or  varieties. 

The  range  of  variation  in  shape  and  size,  especially  of  the  spores, 
under  given  conditions  of  culture  medium  and  temperature,  is  now  used 
only  in  conjunction  with  the  reactions  brought  about  in  various  solu- 
tions to  distinguish  the  various  forms. 

The  true  yeasts  are  characterized  by  the  formation  of  endospores 
and  are  classed  with  the  Gymnoascea.  Each  cell  seems  capable,  under 

*  Prepared  by  F.  T.  Bioletti.  A.  Guilliermond  has  furnished  the  sections  on  the  "  Cytology 
of  Yeasts." 

61 


62  MORPHOLOGY  AND    CULTURE    OF   MICROORGANISMS 

favorable  conditions,  of  developing  into  an  ascus.  Many  unsuccessful 
attempts  have  been  made  to  connect  the  true  yeasts  genetically  with 
various  forms  of  fungi  such  as  Mucor,  Ustilago  and  Dematium.  At 
present  they  must  be  considered  as  distinct  species. 

Some  yeasts  have  a  tendency  during  fermentation  to  remain  at  the 
bottom  of  the  liquid;  others  form  a  thick  foamy  layer  on  top.  These 
are  known  respectively  as  bottom  and  top  yeasts.  No  sharp  distinction 
can  be  made  as  there  are  intermediate  forms. 

The  vegetative  reproduction  in  the  genus  Saccharomyces  takes  place 
by  budding,  in  Schizosaccharomyces  by  fission. 


FIG.  43- — Yeast  cell.     (Original.) 

The  extreme  temperatures  for  budding  lie  between  i°  and  47°,  vary- 
ing with  different  species.  The  optimum  temperature  varies  in  the 
same  way  between  25°  and  35°.  The  rate  of  multiplication  under  favor- 
able conditions  will  range  from  one  to  several  hours  for  the  formation  of 
a  new  cell. 

When  young,  vigorous,  well-nourished  cells  are  supplied  with  abun- 
dant air  and  moisture  at  a  comparatively  high  temperature  under  con- 
ditions that  discourage  budding  (lack  of  nutriment)  they  form  endo- 
spores.  These  spores  are  usually  about  half  the  diameter  of  the  mother 
cell  and  from  one  to  eight  or  more  may  occur  in  each  cell.  They  may 
be  formed  by  cells  before  or  after  budding  and  may  even  change  to  asci 
and  form  new  spores.  They  are  generally  spherical  or  slightly  ellip- 
soidal, rarely  kidney-shaped  (S.  marxianus)  or  furnished  with  a  zonal 
ring  (S.  anomalus)  (Fig.  43). 


YEASTS  63 

In  nutrient  solutions  they  swell,  burst  the  mother  cell,  become  free 
and  germinate  by  budding,  usually  producing  vegetative  cells  directly, 
though  occasionally  producing  first  a  short  promycelium  (S.  ludwigii}. 

In  Schizosaccharomyces  octosporus  the  ascus  is  formed  by  the  fusion 
of  two  cells.  Sometimes  in  other  species,  two  or  more  spores  in  one  cell 
will  fuse  before  germination. 

Staining  with  warm  carbol-fuchsin  and  partial  decolorization  with 
weak  acetic  acid  leaves  the  spores  red  and  the  cell  colorless. 


o  0 


FIG.  44. — Spore-bearing  cells.     A,  S.  pasteurianus.     (After  Bioletti.)   B,  Sch. 
octosporus.     (After  Schionning.}     C,  S.  anomalus.  (After  Kayser.) 


CYTOLOGY  or  YEASTS* 


GENERAL  STRUCTURE  OF  YEASTS. — The  structure  of  yeasts  in  no 
way  differs  from  that  of  the  other  fungi,  only  it  is  seemingly  more  complex 
and  consequently  more  difficult  to  interpret  on  account  of  the  abundance 
of  the  stainable  granulations  which  sometimes  accumulate  in  the  cells 
and  occasionally  hinder  the  differentiation  of  the  nucleus.  This  explains 
why  it  has  until  recently  remained  a  subject  of  controversy.  It  is  now 
fairly  well  understood. 

•  Prepared  by  A.  Guilliermond. 


64  MORPHOLOGY   AND   CULTURE    OF   MICROORGANISMS 

In  order  to  understand  clearly  this  structure,  one  must  observe 

young  cells  taken  from  a  culture  at  the  beginning  of  development. 

For  this  purpose  we  use  Saccharomyces  cerevisia  which,  because  of  the 

relatively  large  size  of  its  cells,  lends  itself  better  than 

any  other  yeast  to  a  cytological  study.     Examined  in 

*  * '  the  living  state,  highly  magnified,   the  cells  of  this 

yeast  show  a  dense  and  homogeneous  cytoplasm  with 

a  group  of  small  vacuoles  or  a  single  large  vacuole  at 

FIG.  45.— Sac-     the  center.     In  the  vacuoles  and  also  in  the  perivacu- 

visi^myCeSyCere~    °^ar  cytoplasm,  we  can  clearly  distinguish  a  great 

cells  examined  in     many  small  shining  granules,  of  varying  sizes,  which 

the  living  state     manifest  Brownian  motion.     It  is  easy  to  stain  them 

in  a  solution  of  J 

neutral  red.  The     in  the  living  state  (Fig.  45)  with  a  very  dilute  solu- 

vacuoles,  stained     tion  of  neutral  red  or  methylene  blue.     These  are 

pale  red,  contain 

m  e  t  a  c  hromatic     only  metachromatic  corpuscles. 

col-  jn  fixed  and  stained  preparations  (Fig.  46,  i-io)  is 
seen  in  each  cell  a  single,  large  nucleus,  whose  struc- 
ture is  exactly  like  that  which  we  have  discussed  in  molds.  This 
nucleus  is  surrounded  by  a  membrane  and  contains  a  hyaline  nucleo- 


A  /^\2 

fQJ  -,    5  10  ... 

*>  *>         -'9  ':*} 

3         4  7    --  ~  '• 

9 


''        u  /^, 

^}(ik  fe          ^ 
''  •  ' 


6          7       8        12  _ 

FIG.  46.  FIG.  47. 

FIG.  46.— Saccharomyces  cerevisia.  i-io,  Young  cells  with  nucleus,  showing  its 
structure.  6-8,  The  same:  division  of  the  nucleus.  11-13,  Cells  after  twenty-four 
hours  fermentation,  with  a  very  large  glycogenic  vacuole  filled  with  lightly  colored 
grains. 

FIG.  47-— Saccharomyces  cerevisia.  Young  cells  fixed  and  stained  by  a  specia 
method  revealing  in  the  cytoplasm  a  chondrium  consisting  of  rod  mitochondria  and 
granular  mitochondria. 

plasm  in  which  is  easily  seen  a  large  nucleolus  and  some  chromatin 
this  latter  is  scattered  through  the  nucleus,  sometimes  found  in  the 
nucleoplasm  in  the  form  of  a  network,  sometimes  reduced  to  a  num- 


her 


YEASTS 


r  of  granules  smaller  than  the  nucleolus,  and  sometimes  even  found 
gathered  on  the  circumference  of  the  nuclear  membrane. 

The  cytoplasm  is  dense  and  homogeneous.  A  special  technic  has 
recently  enabled  the  demonstration  of  a  chondrium  in  the  cytoplasm. 
This  seems  to  consist  both  of  granular  mitochondria  and  of  more  or 
less  elongated  and  flexible  rod-mitochondria  (Fig.  47). 

The  vacuole  shows  in  its  interior  numerous  metachromatic  corpus- 
cles of  varying  sizes  (Fig.  48).  As  in  molds,  these  corpuscles  appear  not 
only  in  the  vacuole,  but  also  in  the  perivacuolar  cytoplasm;  there  they 
start,  and  are  next  diffused  in  the  vacuole  where  they  finish  their  growth, 
then  dissolve  when  the  need  is  felt.  It  is 
difficult  in  the  case  of  yeasts  to  determine 
their  origin;  nevertheless,  observations 
made  of  fungi  with  larger  cells  than  we 
have  previously  described,  show  that  the 
metachromatic  corpuscles  start  in  the 
midst  of  mitochondrial  elements,  and  it 
seems  certain  that  after  that  the  process 
is  the  same  in  yeasts. 

In  the  cytoplasm  of  yeasts,  also,  have 
been  noted  granulations,  which  can  be 
stained  with  ferric  haematoxylin,  and  which 

have  been  named  basophile  grains;  but  these  formations,  which  are  not 
well  defined,  seem  to  us  to  represent  simply  products  from  the  altera- 
tion of  the  chondrium  under  the  influence  of  imperfect  fixing  agents. 

The  membrane  of  yeasts  is  quite  thick  and  very  distinct.  Its 
chemical  nature  is  still  little  known.  According  to  some  authors,  it 
consists  of  a  cellulose;  others  think  that  it  contains  only  pectose.  Ac- 
cording to  Mangin,  it  is  formed  of  callose.  Finally,  some  authors  have 
thought  they  discerned  chitin. 

The  structure  we  have  just  described  is  found  in  all  the  species 
(Fig.  49),  only  it  is  sometimes  much  less  distinct  because  of  the  smallness 
of  the  cells.  In  the  elongated  yeasts,  and  in  the  cells  composing  the 
mycelial  formation  which  are  encountered  under  some  conditions, 
especially  in  the  films,  the  nucleus  generally  occupies  the  center  of  the 
cell;  it  is  situated  in  a  kind  of  matrix  or  bridge  consisting  of  a  very 
dense  cytoplasm,  while  a  vacuole  filled  with  metachromatic  corpuscles 
occupies  each  of  the  two  extremities  of  the  cell. 


FIG.  48. — Saccharomyces  cere- 
visia,  stained  by  a  method  re- 
vealing both  the  nucleus  and 
the  metachromatic  corpuscles. 


66  MORPHOLOGY   AND    CULTURE   OF   MICROORGANISMS 

Summing  up,  the  elements  of  which  a  yeast  cell  consists  are  a  cyto- 
plasm with  a  chondrium,  a  nucleus  with  clearly  differentiated  structure, 
vacuoles  containing  numerous  metachromatic  corpuscles,  a  membrane 
of  a  nature  not  yet  clearly  defined. 

CYTOLOGICAL  PHENOMENA  DURING  MULTIPLICATION.— During  the 
budding  of  the  yeasts,  cytoplasm  enters  the  young  bud  with  some  chon- 
drium; then,  when  the  bud  has  reached  a  certain  size,  the  cytoplasm 
forms  in  it  a  little  vacuole  in  which  appear 
metachromatic  corpuscles  (Fig.  48,  2-7). 

In  the  course  of  these  phenomena,  the 
nucleus  retains  the  position  which  it  occupied 
in  the  mother  cell  before  the  appearance  of 
the  bud.  Only  when  the  bud  is  quite  large 
does  the  nucleus  begin  to  divide.  It  is  elon- 
gated so  that  one  end  penetrates  the  bud;  the 
nucleus  then  resembles  an  elongated  dumb- 
bell with  the  larger  head  remaining  in  the 

77FIG\  49-— Saccharomyces     mother  cell  and  the  other,  smaller  head,  in  the 
elhpsoideus.      Young  cells  _.          N 

each  with  nucleus.  bud  (Fig.  46, 6,  7  and  8;  Fig.  48,  2,  7;  Fig.  49). 

Soon  the  part  of  the  dumb-bell  which  is 

stretched  out  breaks  near  the  neck  of  the  bud,  forming  two  nuclei  of 
unequal  size,  at  first  tapering  spherical  in  shape,  and  later  rounded 
off:  one  is  the  nucleus  of  the  mother  cell  and  the  other  that  of  the 
bud.  This  division  is  therefore  effected  by  the  direct  method;  it  is  an 
amitosis.  In  the  Schizosaccharomyces,  where  the  cells  do  not  multiply 
by  budding  as  in  other  yeasts,  but  by  a  transverse  partition,  the 
nuclear  division  is  effected  by  amitosis:  the  nucleus,  situated  in  the 
center  of  the  cell,  elongates  along  the  longitudinal  axis  of  the  cell  and 
resembles  a  dumb-bell,  ending  by  dividing  in  the  middle,  thus  forming 
two  nuclei  of  the  same  size.  Soon  a  transverse  septum  appears  be- 
tween the  two  nuclei, and  separates  the  two  daughter  cells. 

We  have  now  to  note  the  modifications  which  arise  in  the  structure 
of  the  cells  during  the  different  phases  of  development  and  at  the  time 
of  sporulation. 

VARIATION  IN  THE  CELLULAR  STRUCTURE  DURING  DEVELOPMENT. — 
In  the  course  of  development,  especially  during  fermentation,  yeasts 
reveal  cytological  phenomena  which  render  their  structure  more  com- 
plex and  more  difficult  to  interpret.  Let  us  take  for  example  the  study 


YEASTS  67 

of  the  S.  cerevisice.  After  twelve  hours  of  fermentation,  the  meta- 
chromatic  corpuscles  become  more  numerous.  At  the  same  time,  the 
cytoplasm  forms  little  vacuoles  which  contain  no  metachromatic  cor- 
puscles, but  only  glycogen,  easily  detected  by  iodo-iodide  of  potassium. 
These  are  gradually  fused  into  a  single  vacuole,  which  enlarges  much 
and  modifies  materially  the  cell  structure.  The  glycogenic  vacuole, 
increasing,  pushes  back  to  the  periphery  of  the  cell  the  cytoplasm,  the 
vacuoles  with  metachromatic  corpuscles,  and  the  nucleus  whose  chro- 
maticity  increases  and  which  becomes  homogeneous  in  appearance 
(Fig.  46,  n).  After  forty-eight  hours,  moreover,  the  cell  is  found  to 
consist  of  an  enormous  vacuole  filled  with  glycogen  which  occupies 
most  of  it,  while  the  nucleus,  the  vacuoles  with  metachromatic  cor- 
puscles and  the  cytoplasm  are  pushed  back  to  one  side  of  the  cell,  which 
is  then  transformed  into  a  kind  of  glycogen  sack  (Fig.  46,  12  and  13; 
48,  6-8).  At  this  time  the  glycogenic  vacuole  contains  a  great  many 
small  granulations  (Fig.  46,  12-13),  which  easily  fix  some  staining 
materials,  especially  ferric  haematoxylin,  and  whose  origin  and  signifi- 
cance have  not  been  determined. 

Toward  the  end  of  fermentation,  the  glycogen  gradually  diminishes 
and  the  glycogenic  vacuole  is  gradually  reduced,  then  ends  by  dis- 
appearing. The  cell  after  this  resumes  its  original  structure. 

In  the  course  of  these  phenomena,  the  membrane  apparently  shows 
no  modification.  It  is  known,  however,  that  under  some  conditions, 
yeasts  secrete  gelatinous  substances  which  englobe  their  cells  in  a  kind  of 
jelly  and  so  appear  like  zooglcea  (Hansen).  It  is  well  to  add,  on  the 
other  hand,  that  many  pathogenic  yeasts,  when  living  in  the  host,  have 
the  ability  to  protect  their  cells  against  the  reaction  of  the  organisms, 
by  secreting  a  very  thick  capsule  of  gelatinous  nature:  each  of  their 
cells  is  then  surrounded  by  a  large  capsule. 

CYTOLOGICAL  PHENOMENA  OF  THE  SPORULATION  AND  GERMINATION 
OF  ASCOSPORES. — For  a  study  of  the  sporulation,  we  will  consider  a 
representative  of  the  species  Schizosaccharomyces,  the  Sch.  octosporus, 
in  which  these  phenomena  are  easily  observed  and  especially  well 
understood. 

We  know  that  in  this  yeast,  as  in  some  others,  sporulation  is  pre- 
ceded by  a  sexual  phenomenon  consisting  of  an  isogamous  copulation. 
The  ascus  results  from  the  fusion  of  two  similar  cells.  The  gametes  are 
ordinary  cells  which  have  the  structure  which  we  have  previously 


68 


MORPHOLOGY   AND    CULTURE    OF   MICROORGANISMS 


^T) 

*   b 


^ 


&  •  - 


J 


FIG.  50. — Successive  stages  of 
copulation  and  sporulation  in  Schizo- 
saccharomyces  octosporus. 


described,  with  one  nucleus  and  one  or  more  metachromatic  vacuoles 
containing  corpuscles  (Fig.  50,  a) .  Fusion  takes  place  between  the  two 
cells  which  are  nearest  together.  Each  of  these  two  cells  sends  out  a 
tiny  beak;  the  two  little  beaks  thus  formed  anastomose  and  form  a 

channel  of  copulation  joining  the  two 
cells  (Fig.  50,  b,  c,  d).  The  septum 
separating  the  two  gametes  in  the 
middle  of  the  channel  is  quickly 
absorbed,  and  the  two  cells  then 
have  free  communication.  The  cyto- 
plasm of  the  two  cells  draws  together 
and  mingles  in  the  channel;  there  the 
two  nuclei  draw  near  to  each  other 
(Fig.  50,  e)  and  fuse  into  a  single 
nucleus  (Fig.  50,  /,  g,  h).  Next  the 

zygote  ends  its  fusion;  instead  of  its  original  dumb-bell  appearance,  it 
assumes  the  form  of  an  oval  cell,  then  grows  large  (Fig.  50,  i).  Occa- 
sionally, however,  it  retains  a  vestige  of  the  individuality  of  the  two 
gametes,  showing  two  swellings  joined  by  a  somewhat  narrower  middle 
portion  (Fig.  50,  ;). 

During  this  time,  the  cell  becomes  filled  with  little  vacuoles  and 
assumes  a  more  or  less  alveolar  structure. 
These  vacuoles  contain  a  number  of  metachro- 
matic corpuscles.  The  nucleus  which  occupies 
the  center  of  the  zygote  begins  to  divide.  The 
ascus,  containing  sometimes  four,  sometimes 
eight  ascospores  (Fig.  50,7),  will  then  undergo 
two  or  three  successive  divisions,  as  the  case 
may  be.  These  divisions  are  accomplished  by 
karyokinesis  or  mitosis.  In  the  stages  preceding 
nuclear  division,  the  nucleus  is  very  large  and 
shows  a  very  clear  structure  with  a  nucleolus 
and  a  chromatic  reticulum  (Fig.  51,  a).  It 
soon  elongates  and  assumes  a  special  structure. 
Its  membrane  loses  its  clearness,  and  in  the  midst  of  the  nucleoplasm 
an  achromatic  spindle  appears,  ending  at  each  of  its  two  poles  in  a 
very  small  centrosome  and  containing  at  its  center  a  group  of  fine 
granulations  representing  the  equatorial  plate  (Fig.  51,  b  and  c).  The 


FIG.  5 1 . — Schizosac- 
charomyces  octosporus. 
Various  stages  of  the 
nuclear  division  during 
sporulation. 


YEASTS  69 

nucleolus  always  persists  on  one  side  of  the  spindle.  At  a  subsequent 
stage  the  chromatic  granulations  or  chromosomes  are  divided  between 
the  two  poles  of  the  spindle,  the  nucleoplasm  is  mixed  with  cytoplasm, 
then  the  spindle  elongates,  while  the  chromatic  granulations  form  a 
homogeneous  mass  at  the  two  poles  (Fig.  51  d,  e,  g  and  h).  The 
nucleolus  is  quickly  absorbed,  then  the  two  nuclei  are  formed  at  the 
expense  of  the  two  chromatic  masses  (Fig.  51,  /).  To  summarize, 
therefore,  this  division  consists  in  mesomitoses  of  a  primitive  kind, 
which  appear  to  take  place  in  the  interior  of  the  nucleus,  whose  mem- 
brane is  absorbed  only  at  the  end  of  the  phenomenon.  They  show 
the  characteristics  of  the  mesomitoses  which  have  been  described  in 
the  asci  of  the  higher  Ascomycetes. 


, 
•  . 

•  3 

K  • 
,. 


j 


(w  u. 


6 


FIG.  52. — Successive  stages  of  copulation  and  sporulation  in  Schizosaccharomyces 
pombe.  1-2,  Cells  just  as  sporulation  is  about  to  begin.  3-7,  Union  of  the  two 
gametes  and  nuclear  fusion.  8,  Ripe  ascus.  Cellular  fusion  being  incomplete, 
the  ascus  retains  the  shape  of  the  two  cells  joined  by  a  channel  of  copulation. 

When  these  divisions  are  accomplished,  the  nuclei  seem  to  be  scat- 
tered in  the  cell  (Fig.  50,  i) ;  they  are  soon  surrounded  by  a  thin  layer  of 
cytoplasm  which  is  separated  from  the  cytoplasm  by  a  membrane; 
these  are  the  ascospores.  At  first  very  small,  these  gradually  increase 
at  the  expense  of  the  cytoplasm  which  has  not  been  used  in  their  forma- 
tion— in  other  words  epiplasm — then  reach  the  point  where  they  oc- 
cupy the  whole  of  the  ascus,  after  having  absorbed  this  epiplasm  (Fig. 
50,  j.)  The  metachromatic  corpuscles  scattered  in  the  vacuoles  of 
the  epiplasm  disappear  during  these  phenomena,  being  absorbed  by 
the  ascospores.  At  no  time  during  the  development  of  the  ascus  can 
glycogen  be  seen  any  more  than  in  plant  cells,  but  this  is  replaced 
by  an  amyloid  substance  which  is  stained  blue  by  iodo-iodide  of  potas- 
sium. This  substance  impregnates  the  membrane  of  the  ascospores 
and  disappears  during  their  germination,  utilized  as  a  reserve  product. 

In  some  Schizosaccharomyces  or  ordinary  yeasts  which  bud  (zygo- 


70  MORPHOLOGY  AND  CULTURE   OF  MICROORGANISMS 

saccharomyces)  the  ascus  comes  from  an  egg  which  starts  in  a  similar 
manner  (Fig.  52.)  In  some  species,  this  egg  is  formed  by  a  hetero- 
gamous  copulation  between  an  adult  cell  (macrogamete)  and  a  very 
young  cell  which  has  just  separated  from  the  mother  cell  (micro- 
gamete)  (Fig.  53).  On  the  contrary,  in  most  species,  the  ascus  results 
from  the  simple  transformation  of  an  ordinary  cell  without  previous 
copulation.  Whatever  may  be  its  origin,  the  ascus  shows  cytological 
phenomena  quite  similar  to  those  which  have  just  been  described  in 
Sch.  octosporus,  with  mere  differences  of  detail.  Always  in  Schf 


FIG.  53. — Heterogamous  copulation  in  Zygosaccharomyces  chevalieri.  1-3, 
Gametes  sending  out  a  beak  in  anticipation  of  copulation.  4-7,  Micro-  and  macro- 
gametes  joined  by  their  channel  of  copulation.  8,  The  partition  separating  the 
two  gametes  is  absorbed.  9-18,  The  contents  (nucleus  and  cytoplasm)  of  the  micro- 
gamete  enter  the  macrogamete  and  are  fused  with  the  contents  of  the  latter. 
19-21,  Ripe  asci.  22-23,  Freeing  of  the  ascospores  by  rupture  of  the  membrane  of 
the  ascus. 

octosporus  are  seen  only  a  few  metachromatic  corpuscles  in  the  ascus. 
In  most  of  the  other  yeasts,  on  the  contrary,  the  ascus  contains  a  very 
large  number  of  metachromatic  corpuscles,  and  it  is  easier  there  to  fol- 
low the  evolution  of  these  bodies  which  present  interesting  singularities 
clearly  demonstrating  their  role  as  reserve  substances. 

Let  us  observe,  for  example,  the  cytological  phenomena  which  ap- 
pear during  sporulation  in  Saccharomyces  ludwigii.  In  this  yeast, 
which  shows  no  sexuality  in  the  origin  of  the  ascus,  the  cells  which  are 
preparing  to  sporulate  assume  a  finely  vacuolar  structure  (Fig.  54,  8 
and  9)  and  produce  a  large  quantity  of  reserve  products:  metachromatic 
corpuscles,  glycogen  and  fat  globules.  Metachromatic  corpuscles  spring 
up  in  some  vacuoles,  glycogen  in  others;  as  for  the  fat  globules,  they 


YEASTS  71 

are  located  in  the  cytoplasmic  web.  The  nucleus  is  situated  on  one 
side  of  the  cell,  surrounded  by  a  thin  layer  of  very  thick  and  homo- 
geneous cytoplasm  which  is  to  become  the  sporoplasm,  at  whose 
expense  the  ascospo'res  are  formed,  the  remainder — that  is  to  say  the 
vacuolar  cytoplasm — being  destined  to  compose  theepiplasm  or  nourish- 
ing plasm. 

At  a  later  stage,  the  metachromatic  corpuscles  undergo  a  kind  of 
pulverization  transforming  them  into  small  grains,  and  begin  to  dis- 


• 


fc-ffr.  / .  >-<.v.  ••  ,« JX_-A»  •  +*Gv* 

^  m  wW 

V  Jy     •  F    w 

'9        Ho  \7\\      i: 


FIG.  54. — Sporulation  in  Saccharomyces  ludwigli.  Figs,  i  and  7  showing  the 
evolution  of  the  nucleus.  Figs.  8-9,  the  metachromatic  corpuscles,  stained  by  a 
method  permitting  a  differentiation,  except  in  Fig.  8,  are  dissolving,  and  the  sub- 
stance of  the  vacuole  which  contains  them  shows  a  diffuse  metachromatic  coloring 
(here  gray)  like  the  corpuscles. 


solve  in  the  vacuoles  surrounding  them,  the  latter  at  this  time  taking, 
with  aniline  blue  stains,  a  diffuse  red  coloring  similar  to  that  of  the 
metachromatic  corpuscles  (Fig.  54,  9).  At  the  same  time,  the  nucleus 
undergoes  two  successive  divisions,  but  these  have  not  been  discern- 
ible up  to  the  present  time,  because  of  the  density  and  the  strong 
chromaticity  of  the  sporoplasm  surrounding  the  nucleus.  They  are 
manifested  merely  by  the  appearance  of  the  two  daughter  cells  which 
migrate  to  the  two  poles  of  the  cell,  carrying  with  them  a  part  of  the 
sporoplasm,  which  assumes  the  appearance  of  a  dumb-bell  and  whose 


72  MORPHOLOGY  AND   CULTURE  .OF   MICROORGANISMS 

slender  part  ends  by  breaking  (Fig.  54,  2,  3  and  4).  The  cell,  there- 
fore contains  at  this  time  at  each  of  its  poles  a  small  mass  of  sporo- 
plasm  having  first  one,  then  two,  nuclei  (Fig.  54,  5  and  10).  After 
this,  the  sporoplasm  condenses  around  each  of  these  nuclei  (Fig. 
54,  6),  thus  delimiting  at  each  of  the  poles  two  small  ascospores. 

During  these  phenomena,  the  metachromatic  corpuscles  congre- 
gate around  the  ascospores  (Fig.  54,  n  and  12),  then  gradually  dis- 
solve. The  ascospores  constantly  increase  in  size  at  the  expense  of 
the  epiplasm,  which  becomes  disorganized  and  is  reduced  to  a  vacuo- 
lar  liquid  containing  in  suspension  metachromatic  corpuscles,  fat 
globules  and  glycogen.  They  succeed  in  absorbing  entirely  the  epi- 
plasm and  in  occupying  the  whole  of  the  ascus  (Fig.  54,  13  and  14). 
The  metachromatic  corpuscles,  like  the  glycogen  and  the  globules  of 
fat,  are  then  completely  absorbed  by  the  ascospores,  which  indicates 
clearly  that  they,  as  well  as  the  latter  substances,  act  as  reserve  prod- 
ucts. When  the  ascospores  are  ripe,  they  contain  in  their  vacuoles 
metachromatic  coipuscles  (Fig.  54,  14). 


FIG.  55. — Germination  of  ascospores  in  Saccharomyces  ludwigli.  i,  Beginning 
of  the  fusion  of  the  ascospores.  2,  The  ascospores  are  joined  two  by  two  by  a 
channel  of  copulation,  but  their  nuclei  are  not  yet  fused.  3,  The  nuclei  are  fused. 

4,  At  the  left  two  ascospores,  joined,  have  formed  at  the  middle  of  the  channel  of 
copulation  a  bud  which  has  ruptured  the  membrane  of  the  ascus.     At  the  right,  the 
two  ascospores,  joined  by  a  channel  of  copulation  have  not  yet  fused  their  nuclei. 

5,  Formation  of  the  bud  at  the  expense  of  the  two  fused  ascospores.     Two  other 
ascospores  have  not  yet  begun  their  fusion.     6,  The  bud  formed  at  the  channel  of 
copulation  is  already  established  and  separated  from  this  channel  by  a  transverse 
septum. 

In  all  yeasts,  at  the  time  of  budding,  the  ascospores  have  the  appear- 
ance and  structure  of  plant  cells.  Their  germination  does  not  differ 
from  ordinary  plant  multiplication.  In  some  species,  however,  espe- 
cially in  S.  ludwigii,  copulation,  suppressed  at  the  beginning  of  sporula- 


YEASTS  73 

tion,  is  replaced  by  a  compensating  phenomenon  which  intervenes  at 
the  germination  and  consists  in  the  fusion  of  the  ascospores  two  by  two 
(Fig.  55).  The  ascospores  anastomose  at  their  extremities  by  a  chan- 
nel of  copulation  which,  as  soon  as  the  nuclear  fusion  is  accomplished, 
becomes  the  seat  of  a  budding. 

THE  PRINCIPAL  YEASTS  OF  IMPORTANCE  TO  FERMENTATION 
INDUSTRIES* 

TRUE  YEASTS,  SACCHAROMYCETES. — The  various  yeasts  used  in 
brewing  and  some  of  those  used  in  producing  distilling  material  are 
grouped  together  as  S.  ceremsia.  They  are  large  and  round  or  slightly 
oval. 

They  are  divided  into  three  main  groups — the  bottom  yeasts  which 
are  used  in  the  manufacture  of  German  beer,  and  which,  usually,  are 
capable  of  producing  only  a  moderate  amount  of  alcohol;  the  top  yeasts, 
used  in  English  beers  and  compressed  yeast,  capable  of  producing  more 
alcohol,  and  the  distillery  yeasts,  which  have  great  fermentative  power 
and  produce  large  amounts  of  alcohol. 

Many  forms  of  these  yeasts  have  been  described  in  great  detail  by 
Hansen  and  others  but  the  distinctions  are  based  principally  on  physio- 
logical peculiarities  such  as  the  temperature  and  time  limits  of  film  and 
spore  formation,  and  the  character  of  the  fermented  liquids.  The  vari- 
ous forms  seem  to  be  fixed,  and  to  retain  their  characteristics  unchanged 
under  almost  all  forms  of  treatment. 

The  wine  yeasts,  S.  ellipsoideus,  seem  to  be  even  more  diverse  than 
the  beer  yeasts,  but  have  been  less  thoroughly  studied.  They  are  some- 
what smaller  than  the  latter  and  usually  slightly  more  elongated.  They 
form  spores  much  more  abundantly  and  easily  than  the  beer  yeasts 
and  the  cells  in  film  formation  are  often  much  elongated. 

Their  fermentative  power  is  considerable,  some  of  them  being  capa- 
ble of  producing  over  16  per  cent  by  volume  of  alcohol.  W.  V.  Cruess 
has  obtained  21  per  cent  from  a  Burgundy  wine  yeast.  They  differ  in 
the  flavors  and  aromas  which  they  produce  in  the  fermented  liquid,  and 
especially  in  the  rapidity  with  which  they  settle.  Some  yeasts,  such 
as  those  of  Champagne  and  Burgundy,  form  a  compact  sediment  which 
settles  quickly  and  leaves  the  liquid  clear.  .  Others  remain  suspended 
for  a  long  time  and  settle  with  difficulty.  •  « 

*  Prepared  by  P.  T.  Bioletti.' 


74 


MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 


Every  region  seems  to  have  its  own  forms  and  the  characteristics  of 
the  various  forms  seem  to  be  as  well  fixed  as  those  of  beer  yeasts. 

Wines  are  manufactured  by  the  use  of  these  yeasts.  They  are  also 
employed  in  distilleries.  In  breweries  they  are  considered  disease  yeasts 
and  have  a  deleterious  effect  on  the  beer. 


B 


FIG.  56.— Wine  and  beer  yeasts.  A,  S.  ellipsoideus,  young  and  vigorous;  B,  S. 
ellipsoideus,  (i)  old,  (2)  dead;  C,  S.  cerevisice,  bottom  yeast;  D,  S.  cerevisice,  top  yeast. 
(Original.) 

S.  pyriformis  resembles  in  shape  S.  ellipsoideus,  and  in  association 
with  Bacterium  vermiforme  produces  ginger  beer. 

S.  wrdermanni  is  concerned  in  the  manufacture  of  arrack.  It  fer- 
ments the  sugar  produced  from  rice  by  the  molds,  Mucor  oryzcs  and  Rhi- 
zopus  oryza. 

S.  fragilis  and  other  yeasts  have  been  found  in  kefir  and  other  fer- 
mented Brinks  made  from  milk.  These  yeasts  working  in  conjunction 
with  bacteria  produce  alcoholic  acid  beverages. 


YEASTS  75 

Many  true  yeasts  are  more  or  less  injurious.  They  do  not,  like 
bacteria  and  pseudo-yeasts,  cause  serious  diseases,  capable  of  completely 
ruining  the  fermented  product,  but  they  may  injure  the  quality  more  or 
less.  Some  yeasts  are  useful  in  certain  cases  and  injurious  in  others. 
If  beer  yeasts  become  contaminated  with  wine  yeast  the  resulting  beer 
may  be  persistently  turbid.  If  one  attempts  to  ferment  grapes 
with  beer  yeast,  a  wine  with  a  disagreeable  beer  aroma  and  of  poor 
keeping  qualities  is  produced. 

S.  pasteurianus  occurs  in  several  forms  as  an  injurious  yeast  in  brew- 
eries, causing  bitterness  and  turbidity.  Similar  forms  occur  in  wine  but 
do  little  harm  except  in  the  absence  of  the  true  wine  yeast.  The  cells  of 
this  species  vary  from  oval  to  long  ellipsoidal,  often  being  much  elon- 
gated and  in  film  formation  sometimes  producing  a  branching  mycelium. 
Spores  are  formed  easily  and  abundantly. 

The  apiculate  yeast,  S.  apiculatus,  is  very  abundant  on  grapes  and 
most  acid  fruits.  It  is  very  variable  and  undoubtedly  includes  many 
varieties.  The  cells  are  small,  vary  in  shape  from  oval  to  cylindrical, 
most  of  them  having  an  apiculation  at  one  or  both  ends,  making  them 
pear  or  lemon  shaped.  According  to  Lindner  they  form  spores  in  drop 
cultures,  one  in  a  cell.  Under  favorable  conditions  this  yeast  increases 
with  great  rapidity,  but  is  checked  by  3  to  5  per  cent  of  alcohol.  It 
causes  cloudiness  in  wine,  interferes  with  the  growth  of  the  proper 
yeast  and  injures  the  flavor. 

Many  yeasts,  mostly  small  and  some  of  them  rose-colored,  have 
been  found  on  grapes  and  in  wine,  but  they  do  not  develop  under 
ordinary  conditions  of  wine  making  sufficiently  to  be  harmful. 

Schizosaccharomyces  pombe  is  a  yeast  found  in  pombe  or  millet  beer, 
made  by  negroes  in  Africa.  It  is  cylindrical  and  large,  though  variable 
in  size.  Both  ends  are  rounded.  It  multiplies  by  forming  a  septum 
near  one  end,  the  smaller  division  then  growing  into  a  normal  cell. 
From  one  to  four  spores  are  formed  in  a  cell.  These  spores  are  often 
produced  in  the  fermenting  liquid.  The  fermentative  power  is  high  and 
a  large  percentage  of  alcohol  may  be  formed. 

Several  other  species  of  this  genus  have  been  isolated  from  grapes 
and  from  Jamaica  rum. 

PSEUDO  YEASTS. — Budding  cells  often  occur  in  fermenting  liquids 
which  have  all  the  characteristics  of  yeast  except  that  of  producing 
endospores.  They  are  grouped  together  under  the  name  of  Torula. 


76  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

They  are  usually  small,  spherical  or  slightly  elongated.  Some  species 
produce  a  little  alcohol  and  some  none.  They  seldom  occur  in  suf- 
ficient quantities  to  be  harmful  and  one  form  is  accredited  with  pro- 
ducing the  special  flavor  of  some  English  beers. 

The  forms  included  under  Mycoderma  resemble  yeast  in  shape 
but  produce  little  or  no  alcohol,  are  strongly  aerobic  and  do  not 
produce  endospores.  Their  most  noticeable  characteristic  is  that  they 
grow  only  on  the  surface  of  the  liquid,  where  they  produce  a  thick  film. 
They  cause  complete  combustion  of  the  alcohol  and  other  organic 
matters,  making  beer  and  wine  vapid  and  finally  spoiling  them. 

CULTURE  OF  YEASTS 

PURE  CULTURES. — Yeast  can  be  properly  studied  only  in  pure  cultures.  The 
media  used  are  either  the  liquids  in  which  the  yeasts  are  to  be  used  such  as  wort,  cider, 
grape  juice,  or  a  special  medium  devised  for  a  special  investigation.  An  example  of 
the  latter  is  Laurent's  medium: 

Ammonium  sulphate,  4.yig. 

Potassium  phosphate,  o .  75  g. 

Magnesium  sulphate,  o .  10  g. 

Water,  i  L. 

To  this  is  to  be  added  any  carbohydrate  to  be  studied.  Media  may  be  made 
solid  by  the  addition  of  gelatin  or  agar. 

Pure  cultures  can  be  made,  rarely,  by  inoculation  from  a  naturally  pure  source, 
such  as  the  sporangium  of  a  Mucor. 

Physiological  Separation. — The  first  attempts  at  purifying  mixed  cultures  were  by 
means  of  physiological  differences.  Pasteur  freed  yeast  from  bacteria  by  growing  it 
in  a  medium  containing  2  per  cent,  of  tartaric  acid.  Effront  used  fluorides  in  the  same 
way.  These  methods  may  be  made  more  effective  by  repeated  transfers  of  the 
culture.  Each  transfer  will  contain  a  larger  proportion  of  the  form  most  suited  to 
the  conditions,  until  finally  a  pure  culture  may  be  obtained.  The  principle  of  these 
methods  is  of  great  use  in  practical  fermentation,  but  is  of  little  use  in  rigidly  separat- 
ing forms.  Methods  of  general  application  for  the  latter  purpose  must  be  such  that 
a  single  cell  can  be  isolated  in  a  sterile  medium  and  a  culture  propagated  from 
this  single  cell. 

Separation  by  Dilution  in  Liquid  Media. — A  mixed  culture  is  diluted  with  steri- 
lized water  until  on  the  average  every  two  drops  contain  one  cell.  A  large  number 
of  flasks  of  a  sterilized  nutrient  medium  is  then  inoculated  from  the  dilution,  one 
drop  in  each  flask.  If  the  dilution  has  been  properly  made,  about  half  of  the  flasks 
will  remain  sterile  and  half  will  show  growth.  Many  or  most  of  the  latter  will 
contain  pure  cultures. 

Separation  by  Dilution  in  Solid  Media. — If  we  dip  a  sterilized  platinum  wire  into 
a  mixed  culture  and  then  draw  it  repeatedly  over  the  surface  of  a  solid  culture  medium 


YEASTS 


77 


such  as  a  slice  of  sterilized  potato  or  a  layer  of  nutrient  gelatin  in  a  petri  dish  we  will 
get  a  series  of  streak  cultures.  The  first  of  these  will  develop  a  strong  growth  of  mixed 
forms.  The  last  will  show  more  and  more  isolated  colonies  until  some  of  them  will 
show  only  a  few,  some  of  which  may  be  pure  cultures. 


B 


B 


FIG.  57.— Wild  and  pseudo  yeasts.  A,  S.  pombe.  (After  Lindner).  B,  Torulce. 
(After  Pasteur.}  C,  Mucor,  (i)  spores;  (2)  germinating  spores  and  mycelium.  D, 
S.  apiculatus.  E,  Mycoderma  vini.  (After  Bioletti.) 

The  most  useful  method  of  separation  and  one  which  is  applicable  to  most  cases 
is  that  of  plate  cultures,  first  used  by  Koch  and  improved  by  others.  In  this  method  a 
drop  of  the  mixed  culture  is  thoroughly  distributed  in  10  to  20  c.c.  of  liquefied 
nutrient  gelatin  or  agar.  A  drop  of  this  mixture  is  then  diluted  in  the  same  way  in 
another  portion  of  the  same  medium.  This  process  is  continued  until  the  requisite 


7 8  MORPHOLOGY  AND   CULTURE   OF   MICROORGANISMS 

degree  of  dilution  is  obtained.  The  various  portions  of  nutrient  gelatin  are  then 
poured,  with  precautions  against  outside  infection,  on  glass  plates  or  more  conven- 
iently into  petri  dishes.  On  cooling  and  solidifying,  the  gelatin  imprisons  every  cell, 
each  of  which  on  growing  gives  rise  to  a  colony.  It  has  been  found  that  in  practice 
a  small  percentage  of  these  colonies  may  arise  from  two  adhering  cells  and  thus  fail 
to  be  pure  culture. 

Hansen's  modification  of  the  method  is  intended  to  obviate  this  uncertainty.  By 
making  the  dilutions  in  the  way  described  for  liquid  media,  a  drop  of  gelatin  contain- 
ing only  one  cell  is  obtained,  placed  on  a  cover-glass  over  a  culture  slide  and,  by  direct 
observation,  the  presence  of  a  single  cell  verified.  The  development  and  multiplica- 
tion of  this  cell  can  be  watched. 

DIFFERENTIATION  OF  YEASTS. — With  magnifications  of  300  to  500,  yeast  cells 
can  be  examined  conveniently.  Contamination  with  bacteria  and  molds  of  special 
form  can  be  detected,  but  otherwise  a  simple  microscopic  examination  is  of  little 
value  in  determining  the  purity  of  a  culture.  Some  information  regarding  the 
health,  nutrition  and  vitality  of  the  yeast  may  be  obtained  and  the  form  of  the  spores 
is  of  some  value  in  distinguishing  species.  Yeast  cells  vary  in  size  as  much  as  in 
form  but  under  standard  conditions  each  variety  will  show  a  certain  normal  range  of 
dimensions. 

If  a  young,  vigorous  yeast,  in  a  favorable  liquid  culture  medium,  is  allowed  to 
remain  at  rest  at  a  suitable  temperature  with  full  access  to  air  and  protection  from 
contamination,  a  growth  of  cells  on  the  surface  will  usually  take  place.  This  growth 
may  extend  over  the  whole  surface  (Him  formation)  or  may  be  restricted  to  the  edges 
(ring  formation) .  This  growth  occurs  at  once  with  a  few  species  (S.  membrancefaciens) 
or  at  the  end  of  several  days  (S.  ellipsoideus  II}  or  may  require  several  weeks. 
The  time  and  optimum  temperature  of  film  formation  have  been  used  as  descriptive 
characters. 

All  the  morphological  and  cultural  characteristics  of  yeast  are  insufficient  for 
diagnostic  purposes  and  must  be  supplemented  by  the  physiological  characteristics 
such  as  their  action  on  various  sugars  and  other  carbohydrates. 


CHAPTER  IV 
BACTERIA* 

The  bacteria  naturally  fall  into  quite  distinct  groups  or  orders — 
the  true  bacteria  and  the  sulphur  bacteria. 

A  portion  of  the  true  or  Eubacteria  together  with  the  sulphur  forms, 
are  designated  as  the  higher  bacteria.  The  forms  usually  spoken  of 
as  bacteria  belong  to  the  group  of  lower  bacteria,  and  when  the 
word  "bacteria"  alone  is  used  reference  is  usually  made  to  the  lower 
bacteria.  These  constitute  a  group  of  microorganisms  quite  distinct 
and  characteristic,  while  the  higher  bacteria  form  links,  as  it  were, 
between  the  lower  bacteria  and  other  closely  related  microorganisms. 
The  morphology  of  the  two  groups  will  need  to  be  discussed 
separately.  * 

FORMS  OF  LOWER  BACTERIA* 

FUNDAMENTAL  FORM  TYPES. — The  forms  of  bacteria  are  exceed- 
ingly simple.  They  are  either  spheres,  straight  rods,  or  bent  rods 
(spiral).  In  the  spherical  form  they  are  known  as  cocci,  or  micrococci 
(sing,  coccus  or  micrococcus) .  The  straight  rods  are  bacilli  (sing. 
bacillus)  and  the  bent  rods  are  spirilla  (sing,  spirillum). 


** 

FIG.  58. — Types  of  micrococci.     (After  WUliams.) 


tit 


FIG.  59.— Types  of  bacilli.     (After  Williams.) 
•Prepared  by  W.  D.  Frost,  with  cytology  by  A.  Guilliermond. 

79 


8o 


MORPHOLOGY   AND   CULTURE    OF   MICROORGANISMS 


FIG.  60. — Types  of  spirilla.     (After  Williams.) 

GRADATIONS. — The  difference  between  these  fundamental  form 
types  is  frequently  very  slight.  It  becomes  a  very  difficult  matter, 
for  instance,  to  distinguish  at  times  between  the  micrococcus  and  the 
bacillus.  There  is  a  number  of  bacteria,  and  among  them  the  well- 
known  example  of  B.  prodigiosus,  which  are  described  at  one  time  by  one 
investigator  as  micrococci  and  at  another  time,  or,  by  another  inves- 
tigator, as  bacilli.  The  pneumonia  germ  is  also  another  illustration 
of  an  organism  that  occupies  a  dual  position.  Migula  has  suggested 
a  method  of  differentiating  these  which  will  be  discussed  under  a 
later  head.  The  bacilli  pass  almost  imperceptibly  into  the  spirilla. 
The  cholera  bacillus  of  Koch  is  in  reality  a  spirillum. 


FIG.  6 1.— Involution  forms.  Here  are  illustrated  unusual  forms  of  B.  subtilis, 
water  bacteria,  Bact.  aceti,  Bact.  pasteurianum,  bacteroids  in  root  nodules,  Bact. 
tuberculosis,  Bact.  diphtheria.  (After  Fischer  from  Frost  and  McCampbell.) 


BACTERIA  8 1 

INVOLUTION  FORMS.  * — The  forms  of  bacteria  are  quite  constant  under 
normal  conditions,  but  very  frequently  they  show  abnormal  or  bizarre 
shapes.  These  are  known  as  involution  forms  (Fig.  61).  It  is  some- 
times suggested  that  these  involution  forms  represent  another  stage  in 
the  developmental  history  of  the  organism,  and  upon  this  supposition 
certain  bacteria  which  very  regularly  show  these  involution  forms  have 
been  classified  as  belonging  to  a  different  suborder  from  that  in  which 
the  lower  bacteria  are  placed.  The  ordinary  view  of  the  involution 
forms  is,  however,  that  they  are  degeneration  forms,  that  they  cor- 
respond, in  other  words,  to  the  halt  and  maimed  in  society  and  are  to 
be  accounted  for  by  the  fact  that  they  are  deformed  by  their  own  by- 
products. In  fact,  it  is  quite  probable  that  they  are  autogenic.  In- 
volution forms  are  very  likely  to  occur  in  artificial  culture  and  are  much 
more  common  with  some  species  than  with  others.  (See  page  100.) 

SIZE* 

The  bacteria  were  formerly  spoken  of  as  the  smallest  of  living  things, 
but  since  the  recognition  of  the  ultramicroscopic  organisms  it  is  neces- 
sary to  be  somewhat  more  specific  in  characterizing  their  dimensions. 
The  unit  of  measurement  in  microscopy  is  the  micron  (ju),  or  micro- 
millimeter.  This  is  .001  mm.  or  approximately  1/25000  of  an  inch. 
Applying  this  unit  to  the  bacteria  we  find  that  the  micrococci  and  the 
short  diameter  of  the  bacilli  and  spirilla  average  about  i/x.  The  micro- 
cocci  vary  in  diameter  from  a  small  fraction  of  a  micron  to  three  or  four 
microns  in  diameter.  The  bacilli  are  sometimes  very  small,  as  the 
influenza  bacterium  with  a  width  of  o.2/*  and  a  length  of  o.5/x,  and 
sometimes  very  large  as,  for  example,  the  Bact.  anthracis  with  a  width 
of  i.2fj,  and  a  length  of  5.20;*.  The  spirilla  average  about  i.o/*  in 
diameter  but  may  be  as  long  as  30/1-40;*. 

MOTILITY* 

When  bacteria  are  viewed  under  the  microscope  in  a  living  condition 
many  of  them  are  seen  to  move.  This  movement  may  be  one  of  two 
kinds.  In  some  cases  it  is  progressive,  the  individuals  move  about  from 
one  part  of  the  field  of  the  microscope  to  another  and  change  their  rela- 
tive positions.  In  other  cases  the  movement  is  vibratory,  the  bacteria 
move  back  and  forth  and  rotate  but  do  not  progress  or  change  their 
relative  positions  to  any  extent.  This  latter  form  of  movement  is 
known  as  brownian  movement,  because  it  was  first  described  by  Brown. 

•Prepared  by  W.  D.  Frost. 


82  MOEPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

BROWNIAN  MOVEMENT. — This  movement  is  probably  caused  by  the 
impact  of  the  molecules  of  the  suspending  medium  and  for  this  reason 
is  sometimes  called  molecular  movement.  It  is  not  characteristic  of 
bacteria,  or  indeed  of  life,  but  is  shared  by  many  small  microscopical 
objects  when  suspended  in  a  fluid  medium.  Most  beautiful  examples 
of  brownian  movement  can  be  seen  by  suspending  granules  of  India 
ink  or  carmine  and  examining  them  under  the  microscope.  This 
brownian  movement  is  to  be  sharply  differentiated  from  vital  movement 
which  is  possessed  by  some  bacteria. 

VITAL  MOVEMENT. — As  already  indicated,  bacteria  have  the  power 
of  independent  movement  due  to  inherent  vital  power.  Only  a  few  of 
the  micrococci  are  motile,  while  many  of  the  bacilli  and  spirilla  are.  This 
movement  is  a  change  of  position  and  is  caused  by  certain  protoplasmic 
processes  which  these  bacteria  possess,  known  as  cilia  (sing,  cilium)  or 
flagella  (sing,  flagellum).  The  fact  of  motility  or  non-motility  of  an 
organism  is  of  considerable  value  to  the  systematist.  It  is  determined 
by  examination  in  a  hanging  drop.  At  times,  however,  it  varies  so  little 
from  the  brownian  movement  that  it  is  difficult  to  tell  whether  a  par- 
ticular organism  or  culture  does  or  does  not  possess  vital  movement. 
An  opinion  can  be  more  definitely  formed  at  times  if  some  chemical 
producing  an  anaesthetizing  effect  on  the  bacteria  is  introduced  into 
the  examining  medium.  In  case  the  organism  is  actually  motile  its 
movement  will  be  altered  by  the  anaesthetic  but  in  case  it  is  merely  a 
brownian  movement  there  will  be  no  change. 

ORGANS  OF  LOCOMOTION. — The  protoplasmic  threads  referred  to  as 
the  organs  of  locomotion  are  known  as  flagella,  or  cilia.  The  difference 
between  the  cilium  and  flagellum  is  the  fact  that  a  cilium  has  a  simple 
curve  while  a  flagellum  has  a  compound  curve,  like  a  whip  lash.  Most 
of  the  bacteria  possess  flagella  rather  than  cilia.  The  size,  arrange- 
ment, etc.,  of  these  flagella  are  constant  and  characteristic  of  a  par- 
ticular organism.  Their  structure  and  arrangement,  therefore,  will  be 
discussed  later. 

CHARACTER  OF  MOVEMENT. — Different  bacteria  exhibit  different 
kinds  of  movement.  Some  dart  forward  with  great  rapidity,  others 
move  slowly;  some  move  in  straight  lines,  others  wobble,  but  any 
particular  character  is  quite  constant  and  many  of  the  bacteria  may 
be  recognized  by  their  peculiar  movements. 

RATE. — The  rate  at  which  the  bacteria  travel  when  they  possess 
vital  movement  varies  greatly.  Some  of  them  move  very  fast,  others 


BACTERIA  83 

very  slowly.  Many  of  them  appear  to  move  with  wonderful  rapidity. 
Van  Leeuwenhoek,  when  he  first  saw  these  moving  bacteria,  said  that 
they  traveled  with  such  great  rapidity  that  they  tore  through  one 
another,  but  it  must  be  borne  in  mind  that  under  the  high  powers  of 
the  microscope  the  rate  of  movement  is  magnified  to  the  same  extent 
as  the  object,  and  that  in  reality  the  rate  of  movement  is  not  excessive. 
When  compared  to  their  size,  the  rate  of  movement  is  probably  little 
greater  than  that  of  a  trotting  horse  and  considerably  less  than  that 
of  a  speeding  automobile  or  a  railroad  train. 

REPRODUCTION* 

Reproduction  among  the  bacteria  is  largely  asexual  and  takes  place 
ordinarily  by  what  is  known  as  binary  fission.     In  addition  to  this  a 


QQGDOD 

FIG.  62. — The  division  of  bacterial  cells  (diagrammatic).     (After  Novy.) 

number  of  bacteria  go  into  a  resting  stage,  or  produce  spores.  The 
spore  formation  is  not,  however,  a  method  of  multiplication,  because 
usually  only  a  single  spore  is  formed  in  a  cell,  but  serves  to  tide  the 
organism  through  unfavorable  conditions. 

VEGETATIVE  MULTIPLICATION. — This  is  accomplished  by  means  of 
binary  fission  (Fig.  62).  When  a  bacterium  has  reached  maturity,  fis- 
sion begins.  Division  begins  by  an  invagination  of  the  protoplasm 
in  the  middle  of  the  cell,  which  proceeds  until  the  cell  protoplasm  is 
completely  separated.  The  cell  wall  then  grows  in  and  finally  splits 
forming  the  two  ends  of  the  new  cells.  These  new  cell  walls  are  formed 
at  right  angles  with  the  long  axis  of  the  cell  in  the  case  of  the  bacilli 
and  spirilla,  except  in  rare  instances.  In  the  case  of  micrococci,  the 
throwing  of  the  cell  wall  across  one  diameter  is  quite  as  economical 
as  any  other  and  may  therefore  proceed  in  any  direction.  Migula 
makes  a  considerable  point  of  the  fact  that  bacilli  and  spirilla  elon- 
gate before  division  and  micrococci  divide  before  they  elongate;  this 

•Preparedly  W.  D.  Frost. 


84  MORPHOLOGY  AND    CULTURE    OP   MICROORGANISMS 

would  be  the  criterion  which  he  would  use  to  separate  these  two  form 
types.  A  generation  among  the  bacteria  is  from  one  division  of  the 
cell  to  another.  This  is  sometimes  very  short,  in  fact,  only  twenty  to 
thirty  minutes.  Many  of  the  bacteria  after  half-an-hour's  time  have 
grown  from  newly  formed  cells  to  maturity  and  are  ready  to  divide 
again.  This  makes  it  possible  for  bacteria  to  multiply  with  very  great 
rapidity,  and  if  we  know  the  length  of  the  generation  in  a  particular 
bacterium  it  would  be  easy  enough  to  estimate  the  rate  of  multiplica- 
tion, at  least  theoretically.  It  would  be  only  a  matter  of  geometrical 
progression.  It  is  of  course  quite  impossible  for  the  bacteria  to  main- 
tain their  theoretical  rate  of  growth  for  any  length  of  time,  but,  prac- 
tically, they  grow  with  enormous  rapidity,  as  is  shown  in  cultures  and 
by  the  changes  which  they  bring  about  in  nature,  such  as  the  produc- 
tion of  fermentation  and  the  generation  of  toxin.  Four  periods  in  the 
life  history  have  been  described.  A  latent  or  lag  period,  which  is  the 
time  elapsing  between  the  seeding.and  the  time  at  which  the  maximum 
rate  of  growth  begins;  the  logarithmic  period  or  the  time  when  the  rate 
of  growth  is  at  its" maximum;  a  stationary  period  when  the  increase 
becomes  less  and  less  and  finally  ceases;  and  the  period  of  decline  when 
the  organisms  begin  to  die. 

SPORE  FORMATION  . — A  considerable  number  of  bacteria  form  spores 
within  the  cell.  Because  they  are  formed  within  the  cell  they  are 
spoken  of  as  endospores.  Endospores  are  formed  by  the  bacilli  and  the 
spirilla,  but  not  by  the  micrococci.  Their  chief  value  to  the  cell  is  their 
ability  to  resist  unusual  conditions,  and  to  enable  the  individuals  of  a 
species  to  pass  through  unfavorable  conditions  which  to  the  ordinary 
vegetative  form  of  the  cell  would  prove  disastrous.  At  the  maturity 
of  the  cell,  spore  formation  may  begin.  It  is  an  open  question  whether 
spore  formation  occurs  as  a  regular  stage  in  the  life  history  of  an 
organism,  or  is  produced  only  under  the  stimulus  of  unfavorable  en- 
vironmental conditions.  Both  theories  have  their  advocates.  The 
first  evidence  of  spore  formation  in  the  cell  is  a  granulation  of  the 
protoplasm  of  the  cell.  As  spore  formation  proceeds  the  granules 
become  larger  and  collect  at  one  portion  of  the  cell.  These  granules 
then  fuse  to  form  the  spore,  which  soon  surrounds  itself  with  a  spore 
wall.  At  times  the  spore  is  smaller  than  the  mother  cell  and  is  formed 
without  changing  the  shape  of  the  cell.  At  other  times  it  is  larger 
than  the  mother  cell  and  causes  a  bulging  of  the  latter.  The  position 


BACTERIA  85 


the  spore  in  the  cell  varies  (Fig.  64).  In  some  species  it  is  equatorial, 
in  others  it  is  polar,  and  in  still  others  it  has  an  intermediate  position 
between  equatorial  and  polar.  When  the  spore  is  larger  than  the 
mother  cell  and  is  situated  equatorially  it  causes  the  cell  to  bulge  with 
the  formation  of  a  barrel-shaped  organism,  a  dostridium.  If  the 
spore  is  situated  at  the  poles  and  is  larger  than  the  mother  cell,  a 
capitate  or  drum-stick  bacillus  is  produced.  When  the  spore  is  smaller 
than  the  mother  cell  and  the  cells  form  in  chains,  there  is  frequently  a 
tendency  for  the  spores  to  be  formed  in  opposite  ends  of  contiguous  cells 
of  the  chain  so  that  they  appear  in  pairs.  The  reason  for  this  is  not 
understood.  When  the  spore  has  reached  maturity,  the  mother  cell 
disintegrates  and  finally  disappears,  leaving  the  endospore  free. 

The  endospores  possess  remarkable  powers  of  resistance  due  to  the 
concentrated  character  of  the  protoplasm,  or  to  the  character  of  the 


C  -  -   31313 


FIG.  63.  FIG.  64. 

FIG.  63. — The  formation  of  spores.  (Afler^  Fischer  from  Frost  and  McCampbell.) 
FIG.  64. — Spores  and  their  location  in  bacterial  cells.  (After  Frost  and  McCampbell.) 

spore  wall.  The  resistance  here  may  be  due  to  the  structure  of  the  wall 
itself  or  to  the  chemical  substances  which  it  contains.  It  is  readily  con- 
ceivable that  the  presence  of  certain  .fatty  acids,  or  higher  alcohols, 
might  give  the  spore  its  remarkable  resistance.  These  spores  are  very 
resistant  to  desiccation;  they  have  been  preserved  in  a  dried  condition 
for  many  years.  They  are  also  very  resistant  to  the  action  of  heat; 
some  forms  are  known  to  withstand  a  temperature  of  boiling  water  for 
as  long  a  time  even  as  sixteen  hours.  They  are  resistant  also  to  chem- 
icals and  the  action  of  sunlight,  although  in  some  cases,  as  pointed 
out  by  Marshall  Ward,  the  very  chemical  substances  which  furnish 
them  the  powers  of  resistance  toward  environmental  factors  may  be 
broken  up  under  the  influence  of  sunlight,  forming  poisons  so  that  the 
spore  is  killed  more  readily  than  the  vegetative  cell  would  be. 


86  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

When  these  spores  are  brought  under  favorable  conditions  of 
moisture,  temperature,  and  food  supply,  they  germinate.  There  are 
several  types  of  germination  (Fig.  65).  In  some  cases  the  spore  wall 
ruptures  at  the  pole  and  the  young  cell  emerges  so  that  its  long  axis  is 
in  the  same  direction  as  the  long  axis  of  the  spore.  In  another  type 
the  spore  ruptures  equatorially  and  the  young  cell  emerges  with  its 
long  axis  at  right  angles  to  the  long  axis  of  the  spore.  In  still  another 
type  the  spore  swells  and  the  young  cell  absorbs  the  wall  of  the  spore. 

In  the  lower  bacteria  only  a  single  spore  is  formed  in  a  cell. 
In  the  case  of  the  higher  bacteria,  however,  a  number  of  spores  may  be 
formed  at  the  distal  end  of  the  filament.  These  are  spoken  of  as 
conidia,  and  possess  properties  similar  to  those  of  the  endospores. 


<SS> 


o   0 


FIG.  65. — Spore  germination,  a,  Direct  conversion  of  a  spore  into  a  bacillus 
without  the  shedding  of  a  spore- wall  (B.  leptosporus) ;  b,  polar  germination  of  Bact. 
anthracis;  c,  equatorial  germination  of  B.  suUttis;  d,  same  of  B.  megatherium;  e,  same 
with  "horse-shoe"  presentation.  (After  Novy.) 

In  some  cultures  of  bacteria,  as  for  example  in  the  micrococci, 
certain  cells  seem  to  be  larger  and  different  from  the  other  cells.  In  a 
streptococcus  filament,  certain  cells  suggest  to  the  observer  the  joint 
spores  of  the  algae  and  have  therefore  been  spoken  of  as  arthrospores  or 
joint  .spores.  There  is,  however,  no  evidence  of  an  experimental 
nature,  which  warrants  the  belief  that  these  cells  are  in  reality  spores, 
and  it  must  be  said  that  at  the  present  time  the  presence  of  arthro- 
spores among  the  bacteria  is  purely  hypothetical. 

CELL  GROUPING* 

Bacteria  rarely  occur  singly  but  usually  in  groups.  These  cell 
aggregates  are  frequently  very  constant  and  quite  characteristic  of  the 
organism  possessing  them.  They  are  of  sufficient  definiteness  and 
constancy  to  be  used  by  the  systematists  in  characterizing  large  groups. 

^Prepared  by  W.  D.  Frost, 


BACTERIA  87 

CELL  AGGREGATES  AMONG  THE  MICROCOCCI. — The  grouping  of 
micrococci  depends  upon  the  plane  of  division  and  also  upon  the  cohe- 
sion of  the  cells.  Since  it  is  quite  as  economical  for  the  micrococcus  to 
divide  in  one  direction  as  another,  it  is  possible  for  a  number  of  different 
cell  groupings  to  occur.  Whatever  the  direction  of  the  dividing  walls, 
it  is  usually  quite  constant;  if  a  particular  species  of  micrococci  has  its 
planes  of  division  parallel,  there  will  be  formed  chains  of  micrococci. 
In  some  cases  the  cohesion  is  slight  and  only  two  cells  remain  attached 
to  each  other,  forming  what  are  ordinarily  known  as  diplococci.  There 
is  a  considerable  number  of  very  well-known  bacteria  that  are  diplo- 
cocci (Fig.  66).  If  the  cohesion  is  stronger,  we  have  chains  of  micro  - 
cocci  or  rosaries  formed  which  are  known  as  streptococci.  Well-known 
and  very  important  bacteria  are  grouped  in  this  way.  In  other  micro- 
cocci  the  cell  wall  is  not  formed  continuously  in  parallel  planes  but  in 


oo 

QO 


FIG.  66. — Division  forms  of  micrococci.  a,  Diplococcus,  perfect  form  with 
flattened  opposed  surface  (gonococcus) ,  lanceolate  form  (pneumococcus);  b,  strepto- 
coccus; c,  consecutive  fission  yielding  a  tetrad;  d,  sarcina  form  resulting  from  division 
of  tetrad  c;  e,  staphylococcus.  (After  Novy.) 

planes  which  alternate  at  right  angles  to  each  other.  In  this  way  cell 
aggregates  occupying  two  dimensions  of  space  are  formed.  These  are 
known  as  tetracocci,  or  merismopedia.  Still  again,  the  planes  of  division 
may  proceed  at  right  angles  to  each  other  in  three  dimensions  of  space. 
In  this  case  packets  are  formed  which  are  known  as  packet  cocci,  or 
sarcina.  Another  group  of  the  micrococci  occurs,  known  as  the  staphy- 
lococciy  so  called  because  they  are  arranged  in  irregular  bunches,  like  a 
bunch  of  grapes.  This  arrangement  may  be  due  to  the  fact  that  these 
micrococci  divide  in  many  different  planes,  or  because  during  the  course 
of  their  growth  their  arrangement  is  changed. 

CELL  AGGREGATES  AMONG  THE  BACILLI. — In  the  case  of  the  bacilli, 
one  diameter  is  usually  considerably  shorter  than  the  other,  so  that 
nature  almost  invariably  throws  the  new  cell  wall  across  the  bacilli 
at  right  angles  to  their  long  axis  (Fig.  67).  There  is,  therefore,  only 
one  arrangement  or  cell  grouping  possible,  and  that  is  end  to  end,  so 


88 


MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 


that  streptobacilli  are  formed.  When  arranged  in  pairs,  the  designa- 
tion is  diplobacilli.  The  length  of  the  chains  appears  to  depend  not 
only  upon  the  cohesion  of  the  bacilli  but  also  upon  the  shape  of  the 


\    ^ 


FIG.  67. — Division  forms  of  bacilli,     a,  Single;  b,  pairs;  c,  in  threads.    (After  Novy.) 

end;  those  which  have  square  ends  frequently  have  very  long  chains, 
while  those  with  rounded  ends  have  short  chains  or  occur  singly. 

A  unique  growth-form  or  cell  aggregate  is  that  due  to  the  post  fission 
movement  of  the  cell  as  described  by  Hill  in  cultures  of  Bact.  diph- 


FIG.  68.— Threads  of  Bact.  anthracis.     (After  Migula.) 

theriae.  On  fission  the  two  daughter  cells  are  not  completely  separated 
but  remain  attached  at  one  place.  This  leads  to  a  movement  similar 
to  the  closing  of  a  jack  knife.  In  this  way  the  two  sister  cells  are 
brought  to  rest  at  an  obtuse,  a  right  or  an  acute  angle  to  each  other. 
They  may  be  even  brought  parallel. 


BACTERIA  89 

CELL  AGGREGATES  AMONG  THE  SPIRILLA.  —  The  same  kind  of 
arrangement  is  maintained  among  the  spirilla. 

ZOOGLCEA.  —  Some  of  the  bacteria  secrete  a  mucilaginous  substance 
which  causes  the  cohesion  of  the  cells  frequently  in  considerable  number. 
This  aggregate  of  cells  may  assume  some  characteristic  appearance  and 
a  great  many  attempts  have  been  made  by  systematists  to  make  use 
of  this  in  differentiating  species.  These  zoogloeic  masses  usually 
assume  the  forms  of  pellicles,  but  their  value  as  diagnostic  features  is  not 
great.  The  formation  of  zooglcea  is  very  frequently  only  a  stage  in 
the  life  history  of  an  organism. 

THE  CYTOLOGY  OF  BACTERIA 

*  The  typical  cell,  such  as  that  of  a  higher  plant  or  animal,  is  made 
up  of  cytoplasm  surrounded  by  a  cell  wall.  The  cytoplasm  contains  a 
nucleus.  There  are  also  frequently  present  other  evidences  of  struc- 
ture in  the  cytoplasm,  such  as  nucleolus,  polar  bodies,  etc.  In  addition 
to  these  there  may  be  appendages,  such  as  the  cilia  or  flagella.  In 
the  case  of  bacterial  cells,  we  find  most  of  these  structures  present, 
such  as  cell  wall,  cytoplasm,  and  appendages. 

GENERAL  CONSIDERATION  OF  CYTOPLASM  AND  NUCLEUS.*  —  The 
cytoplasm  of  the  bacterial  cell  is  similar  to  the  cytoplasm  of  other  cells 
except  that  chemical  analyses  seem  to  show  that  it  contains  a  higher 


FIG.  69.  —  Plasmolytic  changes.     (After  A.  Fischer.}     a,  Cholera  vibrio;  b,  typhoid 
bacillus;  c,  Spirillum  undula.     (From  Novy.) 

percentage  of  nitrogen.  As  viewed  under  the  microscope,  in  either  an 
unstained  or  stained  condition,  it  appears  as  a  homogeneous  mass 
filling  the  entire  cell  and  rarely  showing  any  evidence  of  structure. 
Ordinary  stains,  such  as  are  used  in  animal  and  plant  histology,  fail 
to  reveal  the  presence  of  a  nucleus,  the  whole  cell  being  usually  uni- 
formly stained  with  those  stains  generally  characterized  as  nuclear 
stains.  When  these  stains  are  applied  to  some  bacteria,  particularly 
at  certain  stages  of  their  growth,  certain  parts  stain  more  readily  than 
others,  and  we  get  either  what  is  known  as  a  bi-polar  stain  or  polar 

•  Prepared  by  W.  D.  Frost. 


90  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

granules.  In  the  first  case,  the  ends  of  bacilli  are  stained  more  deeply 
than  the  center  so  that  the  cells  appear  very  much  as  diplococci..  This 
bi-polar  stain  is  characteristic  of  such  organisms  as  the  bacterium  of 
chicken  cholera  or  the  bacterium  of  bubonic  plague.  The  polar 
granules  are  frequently  seen  in  the  diphtheria  bacterium  and  may 
be  located  at  the  poles  and  also  at  the  center.  In  this  germ  and  in 
some  others  it  is  possible,  by  special  staining,  to  give  the  granules  a  dif- 
ferent color  from  the  rest  of  the  organism.  In  this  case  these  bodies  are 
spoken  of  as  metachromatic  granules  which  are  considered  later  under 
"Reserve  Products."  The  presence  of  these  granules  might  possibly 
be  explained  upon  the  theory  that  the  cells  are  plasmolyzed  (Fig.  69). 
As  a  result  of  plasmolysis  the  protoplasm  of  the  cell  is  drawn  away 
from  the  cell  wall  and  concentrated  in  areas  which  would  very  well 
explain  the  appearances.  And  it  seems  likely  also  that  the  methods 
employed  in  staining  might  lead  to  plasmolysis,  but  the  metachromatic 
granules  can  hardly  be  explained  upon  this  supposition. 

The  cytoplasm  of  the  bacterial  cell  is  slightly  refractive.  It  is 
colorless  except  in  a  few  cases  in  which  the  green  coloring  matter,  like 
chlorophyl,  is  present,  as,  for  instance,  Bact.  mride  and  Bad.  chlorinum. 
In  the  purple  sulphur  bacteria,  the  coloring  matter  bacteriopurpurin 
is  present.  The  bacterial  cytoplasm  contains  vacuoles  at  times. 

MINUTE  CONSIDERATIONS  OF  CYTOPLASM  AND  NUCLEUS.* — The 
question  of  the  cytology  of  bacteria  has  long  excited  the  curiosity 
of  biologists.  It  is  indeed. of  great  importance  from  many  points 
of  view.  In  the  first  place,  we  are  interested  to  know  whether 
bacteria  are  ordinary  cells  having  a  nucleus;  or  whether,  as  some 
maintain,  they  lack  entirely  a  nuclear  element  and  are  an  exception 
to  the  rule  elsewhere  established.  Moreover,  the  cytologic  study 
of  bacteria  may  furnish  useful  knowledge  concerning  the  phylogeny 
and  taxonomy  of  these  organisms,  a  matter  not  yet  solved.  Finally, 
we  may  hope  that  it  will  throw  light  upon  some  problems  of  a  physio- 
logical or  pathological  nature. 

Unfortunately  this  study  is  very  delicate,  because  of  the  extreme 
minuteness  of  the  bacterial  cells,  so  that  in  spite  of  the  large  number  of 
researches  which  it  has  incited  in  the  last  twenty-five  years,  it  is  to  this 
day  a  matter  of  controversy. 

At  present  three  theories  are  held  by  authors  relative  to  the  inter- 
pretation of  the  general  structure  of  bacteria.  We  will  examine  these 

•Prepared  by  A.  Guilliermond. 


BACTERIA  QI 

three  theories  one  by  one,  endeavoring  to  determine  which  one,  in  our 
opinion,  seems  most  probable. 

One  of  these  theories  claims  that  bacteria  are  cells  of  very  primitive 
organization  lacking  nucleus  and  consisting  simply  of  cytoplasm  with 
vacuoles.  The  cytoplasm  contains  many  stainable  granulations,  but 
these  represent  products  of  nutrition.  Such  an  opinion  scarcely  accords 
with  our  knowledge  of  the  constitution  of  the  other  Protista,  in  all  of 
which  the  existence  of  a  typical  nucleus,  or  at  least  of  chromatic 
elements  replacing  the  nucleus,  has  been  established.  This  view  has 
not,  therefore,  had  many  supporters. 

Another  theory  maintains  that  bac- 
teria have  a  typical  nucleus  and  are  in 
no  way  structurally  different  from  ordi- 
nary cells.  This  opinion  was  suggested 
by  Arthur  Meyer,  who  claims  to  have 
succeeded  in  differentiating,  in  a  great 
many  bacteria,  granules  which  fix  nu- 
clear stains,  and  of  which  one  or  often 
several  appear  in  a  cell.  These  granules 
he  would  consider  nuclei.  It  seems  to 
be  established,  however,  that  the  ma- 
jority of  the  elements  noted  by  Meyer  FlG  70.—  Bacterium  gammari 
are  not  nuclei,  but  reserve  products  and  a  filamentous  bacterium  from 
4-u  r>  ,-  t  JA\  the  intestine  of  Bryodnlus.  (AJter 

common  among  the  Protista  and  known 


as  metachromatic  corpuscles. 

Vejdowsky's  efforts  have  resulted  in  much  weightier  proofs  in  favor 
of  the  existence  of  a  true  nucleus.  In  the  Bacterium  gammarir  a 
species  discovered  by  him  in  the  sections  of  a  little  fresh  water  crus- 
tacean, Gammarus  zschokkei,  Vejdowsky  has  been  able  to  demonstrate 
in  each  cell  a  typical  nucleus  which  is  always  present.  This  nucleus 
appears  very  clearly;  it  consists  of  a  colorless  nucleoplasm  surrounded 
by  a  membrane  and  containing  karyosomes  (Fig.  70).  The  author  had 
the  good  fortune  to  ascertain  in  several  cases  karyokinetic  representa- 
tions of  the  division  of  this  nucleus  (a,  b,  c).  In  short,  the  presence 
of  this  nucleus  is  indisputable. 

The  same  author  discovered  a  similar  structure  in  a  filamentous 
bacterium  found  in  the  digestive  tract  of  an  Annelida  (Bryodrilus 
ehlersi)  (Fig.  70,  d). 


9 2  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

These  conclusions  are  positive,  but  the  species  observed  by  Vej- 
dowsky  are  not  well-defined  bacteria,  and  may  be  thought  to  belong 
to  the  molds  rather  than  to  the  bacteria.  It  has  also  been  said) 
not  without  reason,  that  Bact.  gammari  might  be  a  yeast  of  the  genus 
Schizosacchromyces  and  that  the  filamentous  bacterium  studied  by 
Vejdowski  seems  to  resemble  a  filamentous  mold. 

However  this  may  be,  one  of  Vejdowsky's  pupils,  Mencl,  has  en- 
deavored to  apply  these  conclusions  to  other  bacteria,  which  are  well- 
defined,  notably  B.  megatherium,  but  has  only  succeeded  in  bringing 
forth  proofs  which  are  much  less  convincing  of  the  existence  of  a  nucleus. 
The  author  strived  to  discover  a  nucleus,  but  this  organ  is  not  constant 
and  does  not  show  the  structure  of  a  true  nucleus. 

Both  Kruis  and  Rayman  have  discovered  a  nucleus  in  different 
bacteria  (B.  myco'ides,  radicosus,  etc.).  This  nucleus  appears  only  in 
very  young  cells;  it  is  not  found  in  older  cells,  and  seems  (like  the  nucleus 

noted  by  Mencl)  to  represent  merely  the 
<H^21^      incipient   transverse  septum  which  fixes 
^^^  2.       stains  well  at  the  beginning  of  its  forma- 
O  tion  and  in  some  ways  resembles  a  nucleus. 

3  4  The  studies  of  Penau,  who  also  endea- 

FIG.    71.— Bacillus  megathe-     vored  to  prove  the  existence  of  a  typical 
rium.     (After  Penau.) 


ful.  In  B.  megatherium,  he  describes  the  following  phases.  In  the 
youngest  cells  he  observes  a  stage  where  the  cytoplasm  is  very  dense 
and  uniformly  stained,  without  a  trace  of  differentiation.  Immediately 
succeeding  is  a  phase  where  the  cytoplasm  becomes  less  chromatic  and  is 
filled  with  vacuoles.  At  this  point  the  author  finds  in  each  cell  a  tiny 
granule  (Fig.  71,  i),  homogeneous  and  easily  stained,  situated  at  one  of 
the  poles  of  the  cell,  very  near  the  membrane.  This  granule  he  con- 
siders to  be  a  nucleus.  Moreover,  in  the  cytoplasmic  web  he  observes 
a  series  of  stainable  granules  connected  by  slender  trabeculae,  thus 
forming  a  kind  of  network  which  he  likens  to  mitochondrial  and  chro- 
midial  formations.  At  the  time  of  sporulation,  Penau  finds  an  in- 
crease in  the  size  of  the  nucleus  (Fig.  71,  2  and  3)  which  changes  to 
a  large  granule;  this  is  soon  surrounded  by  a  membrane  and  becomes 
the  spore  (4),  which  is  therefore  formed  mostly  of  chromatin. 

The  same  author  discovers  a  very  different  structure  in  Bact. 
anthracis.     Here,  after  a  stage  of  undifferentiated  structure  which 


BACTERIA  93 

characterizes  the  youngest  cells,  follows  a  phase  where  the  cytoplasm 
becomes  alveolar.  At  this  time,  at  one  of  the  poles  of  each  cell,  appears 
a  very  large  homogeneous  granule  which  Penau  regards  as  a  nucleus. 
This  nucleus,  however,  has  only  an  ephemeral  existence  and  quickly 
undergoes  a  cytolysis  during  which  it  disintegrates.  The  disintegra- 
tion products  then  impregnate  the  trabeculae  of  the  cytoplasm  and  the 
nucleus  becomes  diffuse.  In  a  last  phase  which  corresponds  to  sporo- 
genesis,  the  chromatin  which  impregnates  the  cytoplasm  is  partly  con- 
densed at  one'  of  the  poles,  where  it  forms  first  a  mass  of  grains,  then  a 
large  granule  which  changes  to  a  spore. 

Nothing  is  less  conclusive  than  these  results,  since  the  author  cannot 
discover  an  homologous  structure  in  the  different  species  which  he 
studies,  and  since  the  nucleus  which  he  describes  is  only  a  transitory 
organ  not  showing  the  distinguishing  characteristics  of  a  nucleus. 

To  prove  the  existence  of  a  nucleus  in  bacteria,  it  is  necessary  to 
show  a  nucleus  with  a  differentiated  structure,  the  constant  presence 
of  the  nucleus,  and  to  follow  the  division  of  this  organ  during  the  cellular 
separation.  So  far  no  one  has  apparently  been  able  to  differentiate 
such  an  organ  in  well-defined  bacteria.  We  must  conclude,  therefore, 
that  with  the  exception  of  the  results  obtained  by  Vejdowsky,  all  ob- 
servations so  far  gathered  in  favor  of  the  existence  of  a  typical  nucleus 
in  bacteria  are  by  no  means  convincing. 

The  third  theory  asserts  the  existence  of  a  diffuse  nucleus  in  bacteria. 
It  was  first  suggested  by  Weigert  and  more  carefully  formulated  by 
Butschli.  This  author  describes  in  a  certain  number  of  Sulpha-bacteria 
of  large  size,  Beggiatoa,  Chromatium,  a  kind  of  central  body  occupying 


FIG.  72. — i.  Chromatium  okenii.  2.  Beggiatoa  alba.  These  two  bacteria  have 
a  central  body  containing  chromatic  grains  and  considered  by  Butschli  as  the 
equivalent  of  a  nucleus.  (After  Butschli.} 

nearly  the  whole  volume  of  the  cell  and  consisting  of  an  alveolar  cyto- 
plasm of  highly  stainable  web,  containing  within  its  knots  numerous 
chromatic  granulations  (Fig.  72).  The  remainder  of  the  cell  consists 


94  MORPHOLOGY  AND   CULTURE   OF   MICROORGANISMS 

of  a  thin  cytoplasmic  layer,  less  easily  stainable,  surrounding  the 
central  body.  Butschli  compares  this  structure  with  the  one  which 
has  been  demonstrated  in  the  Cyanophycea,  and  claims  that  the  central 
body  represents  the  equivalent  of  a  nucleus.  It  would  be  a  sort  of  large 
nucleus  occupying  most  of  the  cell,  not  bounded  by  a  membrane,  and 
scarcely  distinct  from  the  cytoplasm.  This  structure  has  recently  been 
verified  in  Chromatium-  okenii  by  Dangeard.  The  Sulpha-bacteria, 
however,  are  organisms  morphologically  entirely  distinct  from  ordinary 
bacteria,  and  are  apparently  directly  related  to  the  -Cyanophycece. 
Such  a  structure  is  not  found  in  other  bacteria,  in  which  it  is  impossible 
to  demonstrate  a  central  body  and  in  which,  one  must  admit,  the 
nucleus  is  still  more  diffuse. 

To  Schaudinn  we  are  indebted  for  the  most  exact  observations  in 
favor  of  the  theory  of  the  diffuse  nucleus.  He  had  the  good  fortune 
to  discover  in  the  intestine  of  the  cockroach,  Periplaneta  orientalis,  a 
bacillus  of  very  large  size  which  he  named  B.  butschlii.  It  is  the  largest 
bacillus  known  at  present  (4^1  wide),  and  lends  itself  readily,  therefore, 
to  cytological  studies.  His  minute  observations  have  shown  that 
there  is  no  nucleus,  the  cells  enclosing  a  finely  alveolar  cytoplasm, 
whose  net  contains  many  small  grains  which  take  nuclear  stains 
(Fig.  73,  1-6). 

At  the  time  of  sporulation  the  chromatic  grains  increase  in  size 
(Fig.  73,  7-9),  then  gather  at  the  center  of  the  cell  in  a  kind  of  axial 
wreath  (Fig.  73,  10).  The  two  extremities  of  this  wreath  quickly  swell 
with  an  accumulation  of  chromatic  grains  and  form  two  granular 
masses,  one  at  either  pole.  These  two  masses  form  the  beginning  of 
the  two  spores,  for  each  cell  forms  two  spores  (Fig.  73,  n  and  12). 
The  grains  which  compose  these  two  rudiments  then  condense  to  form 
two  large  homogeneous  granules  (Fig.  73,  13)  which  strongly  resemble 
nuclei  and  which  Schaudinn  considers  to  be  such.  Around  these  two 
granules  is  soon  condensed  a  thin  cytoplasmic  zone  which  in  turn  is 
separated  from  the  surrounding  cytoplasm  by  a  membrane  (Fig.  73, 
13).  Henceforth  the  spores  cannot  be  stained  by  ordinary  means 
because  of  the  thickness  of  their  membrane  which  prevents  the  pene- 
tration of  stains  (Fig.  73,  14).  The  granules  of  the  wreath,  which 
join  the  two  rudiments  of  spores,  gradually  disappear  as  well  as 
the  cytoplasm,  while  the  spores  increase  in  size.  Then  the  sporangium 
ends  by  breaking  and  setting  free  the  two  spores.  Germination  con- 


BACTERIA  95 

sists  simply  of  a  swelling  of  the  spore,  then  the  formation  of  a  small  rod 
which  issues  from  the  spore  and  forms  a  septum  for  itself  (Fig.  73,  15 
and  1 6).  As  soon  as  the  spore  germinates,  the  nucleus  ceases  to  exist 
as  a  morphologic  entity;  it  is  scattered  in  the  cytoplasm  in  the  form  of 
little  grains. 


~v    ••-:      •'•::        r. 

Iff  I 


10    II     IZ     13    14 


FIG.  73. — Bacillus  butschlii.  1-16,  Vegetative  cells  and  their  division.  7-9,  Begin- 
ning of  sporulation:  the  cells  about  to  sporulate  are  partitioned  off  crosswise;  then 
the  septum  thus  formed  is  absorbed,  at  which  time  sporulation  begins.  Schaudinn 
considers  this  partitioning  off  followed  by  fusion  of  the  two  daughter  cells  as  a  rudi- 
mentary sexuality.  10-13,  Formation  of  the  beginnings  of  the  two  spores,  at  the 
poles  of  the  cell.  14,  Ripe  spores.  15-16,  Germination  of  the  spore.  (After 
Schaudinn.) 

In  another  bacillus  smaller  in  size  (B.  sporonema),  Schaudinn  has 
found  an  analogous  structure  only  at  the  time  of  sporulation;  he  does 
not  prove  the  formation  of  an  axial  filament  but  only  the  condensation 
of  a  portion  of  the  chromatic  grains  into  a  large  granule  which  forms  the 
beginning  of  the  spore  (Fig.  74). 

By  the  fact  that  in  these  two  bacilli  the  beginning  of  the  spores 
appears  as  a  granule  equivalent  in  some  respects  to  a  nucleus  and 
resulting  from  the  condensation  of  a  portion  of  the  stainable  grains, 
Schaudinn  is  led  to  believe  that  these  grains  are  composed  of  chroma  tin 
and  represent  a  kind  of  diffuse  nucleus. 


96  MORPHOLOGY  AND    CULTURE    OF   MICROORGANISMS 

These  results  have  been  confirmed  by  our  studies  of  a  large  number 
of  endospore  bacilli  (B.  megatherium,  radicosus,  mycoides,  aster ospor us, 
alvei).  Upon  examination  at  the  very  outset  of  their  development, 
these  bacteria  present  a  homogeneous  appearance  and  are  uniformly 


ill 
11® 

I       2       3 


5 

FIG.  74. — Bacillus  sporonema.  i,  Cell  about  to  sporulate.  2,  This  cell  grows 
narrow  at  the  center,  as  if  it  were  going  to  be  divided  (Schaudinn  regards  this  pinch- 
ing together  which  afterward  disappears  (5),  as  the  vestige  of  an  ancestral  sexuality 
like  that  of  B.  biitschlii).  3-5,  Formation  of  the  beginning  of  the  spore.  (After 
Schaudinn.) 

stained  with  no  great  differentiation,  explicable  by  the  density  of  the 
cytoplasm  or  by  a  special  condition  of  the  membrane.  At  this  stage 
the  cells  are  in  the  process  of  active  divisions,  after  which  the  transverse 
septa  are  formed  as  follows:  On  the  side  walls  of  the  bacillus  appear 
two  small  granules  which  take  some  stains  (Fig.  75,  i).  These  soon 


10     II 


FIG.  75.  —  i-io,  Bacillus  radicosus.  i,  Beginning  of  development.  2-3,  Cells 
at  the  end  of  eight  hours;  4-6,  sporulation.  9-10,  Cells  in  which  the  chromatic 
grains  are  located  in  the  middle  in  a  mass  slightly  resembling  a  nucleus.  11-12, 
Spirillum  wlutans. 

disintegrate  at  the  center  of  the  cell  to  form  a  thin  band  marking  out 
the  two  daughter  cells  and  forming  the  beginning  of  the  transverse 
septum.  This  strongly  resembles  a  nucleus  and  has  apparently  been 
considered  as  such  by  a  number  of  authors  (Rayman  and  Krius,  Mencl). 
Toward  the  eighth  hour  of  development,  the  cells  show  clearly  their 


BACTERIA  97 

structure  which  is  changed  in  appearance;  the  cytoplasm  vacuolizes  and 
ends  by  displaying  a  fine  alveolar  structure.  The  web  contains  in  its 
knots  small,  highly  stainable  granules  (Fig.  75,  2  and  3).  In  some 
cases  (cultures  on  special  media  for  example),  there  is  noticeable  a 
localization  of  these  granules  at  the  center  of  each  cell,  forming  a 
granular  region  which  recalls  somewhat  the  appearance  of  a  large 
nucleus  and  which  is  separated  into  two  portions  at  the  time  of  the 
cellular  division  as  if  it  were  indeed  a  true  nucleus  (Fig.  75,  7  and  10). 

These  granules  fix  the  nuclear  stains,  and  it  seems  permissible  to 
consider  them  chromatic  in  nature. 

At  the  time  of  sporulation  there  forms  at  one  of  the  poles  of  the 
cell  a  small  oval  mass,  easily  stained,  which  is  like  a  nucleus  in  appear- 
ance (Fig.  75,  4  and  5).  This  results  from  the  condensation  of  part  of 
the  chromatic  granules  of  the  cytoplasm,  gradually  grows  larger,  and 
changes  to  a  spore.  When  the  spore  has  reached  a  certain  size,  it  is 
surrounded  by  a  membrane  which  prevents  the  penetration  of  ordinary 
stains  (Fig.  75,  6);  it  appears  then  like  a  large  colorless  sphere  in  the 
stained  cytoplasm  of  the  cell  (Fig.  75,  6). 

At  no  stage  of  the  development  have  we  observed  the  least  trace  of 
a  nucleus.  May  there  be  a  nucleus  which  our  present  technic  would 
not  enable  us  to  differentiate?  That  has  seemed  to  us  scarcely  probable, 
for  if  this  nucleus  existed,  it  would  certainly  be  visible  in  a  species 
as  large  as  B.  butschlii  and  would  not  have  escaped  Schaudinn.  The 
most  reasonable  hypothesis,  the  one  which  we  have  adopted,  is  to 
consider  like  Schaudinn  that  bacteria  contain  chromatin  more  or  less 
mingled  with  cytoplasm,  differentiated  in  the  case  of  small  grains  and 
condensing  at  the  time  of  sporulation  to  form  the  spore  which  would 
consist  principally  of  chromatin.  The  cells  of  bacteria  would  accordingly 
have  a  very  primitive  structure. 

Granted  the  clearly  demonstrated  existence  of  this  particular  struc- 
ture in  the  Cyanophyceoe,  there  is  no  reason  for  not  admitting  that  the 
nucleus,  very  rudimentary  in  the  Cyanophycece,  might  be  even  more  so 
in  bacteria,  being  reduced  to  a  diffuse  nucleus  consisting  of  chromatic 
grains  scattered  in  the  cytoplasm. 

These  observations  have,  moreover,  received  a  series  of  new  con- 
firmations by  the  labors  of  a  great  many  authors  (Swellengrebel, 
Ruzicka,  Ambrez,  etc.)  and  especially  by  the  later  researches  of  Dobell. 
The  latter  investigator  discovered,  in  the  intestines  of  frogs  and  toads, 

7 


98  MORPHOLOGY  AND    CULTURE    OF   MICROORGANISMS 

a  large  bacillus  (2^  wide)  almost  as  large  as  B.  butschlii,  and  named  it, 
B.  flexilis.  This  species  shows  exactly  the  same  cytological  charac- 
teristics as  B.  butschlii  (Fig.  76). 

Through  a  study  of  a  number  of  different  bacteria  found  in  the  in- 
testine of  toads,  frogs  and  lizards,  D  obeli  has  endeavored  to  show  that 
this  diffuse  nucleus  is  not  original,  but  derived  from  the  retrogression 
of  a  more  highly  differentiated  nucleus. 

Thus  in  various  micrococci  he  was  able  to  show  in  each  cell  the 
existence  of  a  central  stainable  granule,  dividing  by  constriction  at  the 
time  of  cellular  division,  and  which  he  regards  as  a  nucleus  (Fig.  77, 


IZ 


16 


FIG.  77. 
i,  Beginning  of  the  division  of  a  cell  about  to  sporu- 


I 

4 

FIG.  76. 

FIG.  76. — Bacillus  flexilis. 

late  (vestige  of  sexuality).  2,  Disappearance  of  the  incipient  division.  3,  Forma 
tion  of  the  chromatic  axial  filament.  4,  Formation  of  the  beginning  of  two  spores 
5,  Ripe  spores.  (After  D  obeli  ^ 

FIG.  77. — Various  bacteria,   showing  the  successive  types  of  the  retrogress! 
of  the  original  nucleus  and  its  transformation  to  a  diffuse  nucleus.      (After  DobelL 

1-5).  In  other  cocco-bacillary  species  of  bacteria  characterized 
spherical  shape  capable  of  elongation,  D  obeli  discovers  a  similar  nucleus 
in  the  spherical  cells.  When  the  cell  lengthens  and  assumes  the  ap- 
pearance of  a  bacillus,  this  nucleus  changes  to  a  spiral  axial  filament 
(Fig.  77,  5  and  6). 

In  various  bacilli  the  same  author  demonstrates  a  filament  which  is 
ever  present  (Fig.  77,  7-11).  The  spore  results  from  the  condensation, 
at  one  of  the  poles,  in  the  shape  of  a  large  chromatic  granule,  of  part 
of  the  grains  which  compose  this  filament  (Fig.  77,  12  and  13).  An 
interesting  variation  of  this  structure  is  found  in  B.  saccobrinchi. 


BACTERIA  99 

In  this  bacillus  is  noticed  first  an  initial  stage  where  the  nucleus  is 
represented  by  an  axial  filament  quite  similar  to  that  oiB.spirogyra 
(Fig.  77,  14).  In  the  course  of  development,  however,  this  filament 
resolves  itself  into  a  great  many  grains  which  scatter  through  the 
cell  (Fig.  77,  15  and  16).  The  nucleus  then  becomes  diffuse.  Part  of 
this  diffuse  nucleus  next  condenses  at  the  time  of  sporulation  into  a 
large  chromatic  grain  which  forms  the  beginning  of  the  spore.  Finally, 
in  other  bacilli,  Dobell  finds  in  the  whole  development  no  more  than  a 
diffuse  nucleus,  that  is,  the  structure  described  by  Schaudinn  and  by 
Guilliermond. 

In  the  group  of  spirilla,  Dobell  notices  these  three  types  of  structure: 
In  some  species  he  finds  present  a  spherical  body  resembling  a  nucleus; 
other  species  show  a  zigzag  or  a  spiral  filament;  still  others  have  a 
diffuse  nucleus. 

From  these  observations,  Dobell  feels  authorized  to  conclude  that 
bacteria  are  organisms  originally  containing  a  nucleus,  but  in  which  the 
nucleus,  as  a  result  of  parasitism,  has  undergone  a  series  of  retrogres- 
sions which  have  ended  by  making  it  diffuse. 

This  opinion  would  have  the  advantage  of  reconciling  opposed 
theories.  It  would  explain  how  some  authors  have  been  able  to  dis- 
cern a  true  nucleus  in  various  forms. 

Another  more  weighty  reasoning  which  might  also  explain  these 
contradictions  is  the  fact  that  under  the  name  of  bacteria  are  gathered 
forms  perhaps  very  different,  some  of  which  seem  to  belong  to  the 
Sulpho-bacteria  and  others  might  be  considered  as  molds. 

Although  we  have  just  mentioned  numerous  works,  the  conclusion, 
to  my  mind,  would  be  that  while  some  bacteria  may  contain  a  more  or 
less  rudimentary  nucleus  whose  existence  is  nowhere  else  precisely 
demonstrated,  so  far,  in  the  great  majority  of  the  species,  nothing  more 
has  been  found  than  a  diffuse  nucleus  consisting  only  of  grains  of  chro- 
matin  scattered  through  the  cytoplasm. 

Life  Cycle  of  Bacteria*. — The  life-cycle  of  bacteria  will  prove  a  very 
important  factor  in  the  study  of  their  morphology,  their  cultivation, 
their  cultural  characteristics  and  their  classification,  if  its  development 
takes  place  along  the  line  so  definitely  advanced  by  Lohnis  and  Smith  f. 
The  variation  in  the  appearance  of  a  species  of  bacteria  has  long  been 

*  Prepared  by  the  Editor. 

t  Lohnis,  P.  and  Smith,  N.  R.:  Jour.  Agr.  Research,  VI,  18,  675.  1916. 


100  MORPHOLOGY  AND   CULTURE   OF  MICROORGANISMS 

recognized;  cultivation  has  been  fraught  with  difficulties  which  have  at 
times  been  in  some  way  associated  with  the  change  in  form  or  in  a  sense 
connected  with  " involution"  alterations;  cultural  characteristics  have 
likewise  been  subject  to  variations  which  have  depended  upon  the 
so-called  vigor  of  the  organism;  and  classification  of  bacteria  may  be 
materially  affected  since  some  of  the  cycles  approach  closely  those  of 
protozoa. 

Perhaps  the  most  significant  changes  upon  which  the  life-cycle  of 
bacteria  is  based  may  be  those  represented  by  Jones,*  and  Lohnis  and 
Smith  in  the  life  of  A  zotobacter-types.  The  polymorphous  character  of  the 


FIG.  78. — Change  of  Azotobacter  from  the  normal  cells  (I)  to  arthrospores  (II) 
and  involution  forms  (III)  to  be  lost  in  symplastic  stage  (IV)  and  recovering  cell- 
form  in  V.  Diagrammatic  iroptf  Lohnis  and  Smith. 

Azotobacter  group  has  been  a  matter  of  intense  interest  for  a  long  period. 
Lohnis  and  Smith  have  not  only  endeavored  to  follow  the  variations 
through  a  consistent  historical  developmental  cycle  but  have  attempted 
to  organize  their  observations  and  have  them  in  accord  with  past 
observations. 

The  organism  may  be  assumed  to  exist  in  the  form  of  a  distinct  cell 
and  at  other  times  in  an  amorphous  condition  called  by  the  authors,  the 
symplastic  stage.  In  the  usual  cell-form  the  organism  may  multiply 
by  fission  as  is.  the  case  with  all  bacteria,  may  produce  endospores 

*Jones,  D.  H. :  Cent.  f.  Bact.;  Trans.  Royal  Society  of  Canada,  1913. 


BACTERIA  IOI 

as  is  a  common  mode  of  reproduction,  or  arthrospores,  when  the  entire 
organism  appears  to  transmute  to  a  resting  stage  or  spore,  or,  the  organ- 
ism may  pass  to  the  amorphous  or  symplastic  condition.  There  is 
also  a  possibility  of  a  union  or  "  con  junction"  of  cells  suggesting  the 
functioning  of  gametocytes. 

In  passing  into  the  symplastic  stage  the  cells  passing  through  involu- 
tion forms  appear  to  form  clumps  and  lose  completely  their  individual- 
ity of  form  and  contents  in  a  general  mass  of  disorganized  protoplasmic 
debris.  Presumably  scattered  throughout  this  mass  exists  what  may 
be  recognized  in  protozoal  forms,  yeast  cells,  et  cetera,  nuclear  centers, 
for  out  of  this  more  or  less  homogeneous  unvarying  background  of 
protoplasmic  substance  appear  many  lines  resulting  in  modified  forms 
which  pass  on  to  forms  similar  to  the  original  cellular  forms  from  which 
this  amorphous  mass  was  at  first  derived. 

.  The  form  of  Azotobacter  upon  which  this  life-cycle  theory  is  based 
may  not  be,  of  course,  conclusive;  however,  Jones  has  confirmed  many 
of  the  findings  of  Lohnis  and  Smith  in  the  case  of  Azotobacter  but  is 
not  ready  to  subscribe  to  all  of  their  interpretations.  Jones  *  claims,  too, 
that  so  far  as  other  species  of  bacteria  are  concerned  in  this  theory 
of  life-cycle,  he  has  been  unable  to  confirm  Lohnis  and  Smith  who 
assert  that  in  the  forty-eight  species  studied,  they  find  practically  the 
same  developmental  cycle. 

This  subject  is  of  so  wide  importance  that  it  deserves  much  atten- 
tion and  study. 

RESERVE  PRODUCTS.! — Besides  the  grains  of  chromatin  which  we 
have  just  been  considering  in  bacteria  are  found  other  granulations 
which  do  not  show  the  characteristics  of  chromatin  and  which  act  as 
products  of  nutrition.  These  granulations  are  characterized  by  the 
reddish  color  which  they  assume  with  most  of  the  aniline  blue  or  violet 
dyes,  as  well  as  with  haematoxylin.  These  bodies,  which  are  common 
to  the  majority  of  the  Protista,  are  metachromatic  corpuscles. 

They  are  found  in  larger  or  smaller  numbers  according  to  the  species, 
the  age  of  the  cells,  and  the  medium  in  which  they  are  living.  Some 
bacteria  contain  few  metachromatic  corpuscles  (B.  radicosus,  megathe- 
rium, mycoides);  others  produce  many  (B.  alvei,  asterosporus,  Sp. 
volutans,  Bact.  tuberculosis  and  diphtheria).  The  metachromatic 

Mones,  D.  H.:  Jour,  of  Bact.,  Vol.  V,  p.  325. 
t  Prepared  by  A.  Guilliermond. 


102  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

corpuscles  appear  at  the  beginning  of  development  in  the  form  of  very 
small  grains,  which  generally  increase  gradually  in  size  during  de- 
velopment, and  finally  are  absorbed  in  the  very  old  cells.  They  are 
sometimes  distributed  through  the  whole  cell  (Spirillum  volutans)  as 
grains  of  chroma  tin  (Fig.  79,  8  and  9),  but  most  often  they  tend  to 
gather  at  the  two  poles  of  the  cell,  or  line  up  all  along  the  bacillus 
(Fig.  79,  i  to  4,  6,  10,  n).  In  some  species  (B.  alvei,  asterosporus, 
Bad.  tuberculosis  and  diphtheria),  these  corpuscles  grow  bigger  until 
they  attain  relatively  large  dimensions,  surpassing  the  bacillus  in  size. 

Thus  they  cause  a  series  of  swellings  all 
*;  >; ;.;    *  along  the  bacillus,  which  in  consequence 

*  *  '  $  appears  somewhat  like  a  necklace  (Fig. 
79,  n).  They  then  give  the  illusion  of 
spores;  one  can  easily  understand  the 
error  of  some  authors  who  have  confused 
them  with  spores,  notably  in  the  case  of 
5  9  "10  /%*  the  Bact.  tuberculosis. 

In  B.  asterosporus,  the  metachromatic 
FIG.  70. — Various  bacteria  i  -n  i 

stained   by   a   method   which    corpuscles  usually  appear  in  the  youngest 

differentiates   only    the  meta-  cells,  singly  and  in  the  shape  of  a  small 

chromatic    corpuscles.        1-4,  -,  111  i_v 

Bacillus  radicosus.    5-6,  Bacil-  central  granule  closely  resembling  a  nu- 

lus  asterosporus.    7,  The  same,  cleus  and  which  A.  Meyer  seems  to  have 

The    cells    have  formed   their  ,    •,        f  •>    /••-•  \ 

spore  and  the  metachromatic  taken  for  such  (FlS«  79,  S)- 

corpuscles  outside  of  the  spores          During  sporulation,  the  metachromatic 

have  not  yet  been  absorbed  by  i  •  .    •  ,   •  j       r    ,1 

it.     8-9,    Spirillum   wlutans.  corpuscles  exist  just  outside  of  the  spore 

lo-u,  Bacillus  ahei.  (Fig.  79, 7),  then  are  finally  absorbed  by  it. 

They  therefore  act  like  reserve  products. 

Moreover,  in  the  cells  of  bacteria  other  reserve  products,  notably 
globules  of  fat  and  of  glycogen,  have  been  found. 

BACTERIAL  CELL  WALL. — General  Structure* — All  the  bacteria  have 
cell  walls  and  it  is  these  that  give  definite  form  to  the  cell.  These  walls 
are  rigid  and  elastic  and  are  probably  made  up  of  two  layers,  the  outer  one 
of  which  is  able  to  deliquesce  and  form  capsules,  or  perhaps  zooglosa. 
The  inner  part  retains  the  elasticity  and  gives  the  form  to  the  bacteria. 
These  cell  walls  are  readily  permeable  to  water  and  it  is  through 
them  that  all  of  the  nourishment  of  the  cell  is  obtained;  that  is, 
there  are  no  openings  for  the  entrance  of  food  or  the  discharge  of 

•  Prepared  by  W.  D.  Frost. 


BACTERIA 


103 


by-products,  but  the  intake  and  output  goes  on  through  the  cell  wall 
which  is  entire. 

Minute  Structure  of  Cell  Wall.* — In  some  species  of  large  size, 
the  membrane  can  be  distinguished  when  strongly  magnified,  and 
appears  with  a  double  contour.  Usually  it  is  scarcely  visible,  and  can 
be  observed  only  when  the  contents  of  the  cell  has  been  contracted  by 
plasmolysis  or  by  a  suitable  reagent.  It  is  sometimes  thin,  some- 
times more  or  less  thick.  In  the  latter  case,  it  is  often  possible  to 
recognize  two  layers,  an  inner  or  cuticular  layer,  very  thin  and  trans- 
parent; and  the  other  external,  not  so  well  defined  and  thicker,  jelly- 
like  in  appearance.  This  latter  or  gelatinous  layer  seems  to  result 
from  a  special  differentiation  of  the  peripheral  zones  of  the  inner  layer. 
The  outer  layer  ordinarily  resists  staining  reagents  and  appears  as  a 
kind  of  transparent  zone  about  the  colored  elements.  It  can  acquire 
a  relatively  great  thickness,  and  the  formations  described  as  capsules 
are  only  an  exaggeration  of  this  gelatinous  layer. 

Schaudinn  has  been  able  to  observe  quite  care- 
fully the  construction  of  the  cuticular  layer  in 
B.  butschlii.  According  to  him,  the  membrane 
seen  in  profile  would  appear  to  consist  of  a 
series  of  disks  alternately  clear  and  cloudy  (Fig. 
80,  A  and  B).  Seen  from  the  front,  it  would 
give  the  impression  of  a  network  whose  meshes 
are  more  refringent  and  stain  more  highly  (C). 
It  is  laid  on  a. peripheral  zone  of  cytoplasm,  a 
kind  of  ectoplasm  with  closer  network,  and  is 
clearly  differentiated  from  the  rest  of  the  cyto- 
plasm. The  spore  is  provided  with  a  double 
membrane  and  has  at  one  of  its  poles  a  sort  of 
micropyle  through  which  germination  is  effected 
(Fig.  73,  15  and  16). 

The  chemical  composition  of  the  membrane 

is  little  known.  According  to  some  authors,  this  membrane  consists 
of  cellulose;  according  to  others,  it  contains  a  lipoid  substance; 
finally,  by  many  authors  it  is  supposed  to  be  composed  principally 
of  nitrogenous  compounds.  Let  us  remark  further  that  chitin  has 
supposedly  been  detected  therein. 

*  Prepared  by  A.  Guilliermond. 


FIG.  80. — A  and  B, 
Structure  of  the  mem- 
brane and  of  the  ecto- 
derm  in  Bacillus 
butschlii.  C,  Membrane 
of  the  same  bacillus, 
front  view.  (After 
Schaudinn.} 


IO4  MORPHOLOGY  AND    CULTURE   OF   MICROORGANISMS 

Capsules.* — A  considerable  number  of  the  bacteria  regularly,  or 
under  certain  conditions,  form  what  are  known  as  capsules  (Fig.  81). 
These  "are  mucilaginous  envelopes  which  in  width  frequently  exceed 
that  of  the  organism  itself.  In  microscopical  preparations  of  bacteria 
it  is  important  to  differentiate  these  from  artifacts,  since  by  ordinary 
staining  methods  the  capsules  are  not  colored  but  appear  as  colorless 
areas  surrounding  the  bacteria.  If,  due  to  shrinkage  of  the  bacteria, 
or  other  material  on  the  preparation,  clear  spaces  are  formed,  it  is 
readily  seen  that  these  might  be  confused  with  the  real  capsule.  It  is 


FIG.  81. — Capsules.     Bact.  pneumonia  (Friedlander).     (After  Weichselbaum  from 
Frost  and  McCampbell.) 

possible  to  stain  the  capsules  by  special  methods;  these  must  be  used  in 
order  to  determine  positively  the  existence  of  the  capsules.  The 
bacteria  which  grow  in  the  bodies  of  animals  frequently  contain  these 
capsules  but  fail  to  show  them  when  grown  upon  artificial  culture  media. 
It  is  difficult,  therefore,  to  determine  whether  or  not  an  organism  has  a 
capsule  by  mere  examination  of  cultures.  Some  culture  media,  how- 
ever, do  cause  a  formation  of  capsules  in  the  case  of  capsulated  bacteria. 
These  are  blood  serum,  sometimes,  and  milk,  usually.  Beautiful  cap- 
sules can  be  obtained  by  growing  such  bacteria  as  the  Bact.  pneumonia, 
Bact.  capsulatum,  and  Bact.  Welchii  in  milk  cultures.  Strept.  mesen- 
teroides  is  a  bacterium  which  grows  in  the  syrup  of  the  sugar  refineries 
and  forms  abundant  capsules.  This  organism  changes  the  char- 

*  Prepared  by  W.  D.  Frost, 


BACTERIA 


105 


acter  of  the  syrup,  and  its  entrance  and  growth  is  frequently  the  cause 
of  serious  loss. 

FLAGELLA.  —  General  Consideration  of  Flagella.*  —  The  flagella  are 
very  narrow  thread-like  structures.      It  is  not  known  how  narrow  since 


r 

FIG.  82.  FIG.  83.  FIG.  84. 

FIG.  82.  —  Chromatium  okenii;  2,  Bacterium  lineola;  3,  4  and  5,  sulpho-bacteria; 
7,  Ophidomonasjenensis;  8,  and  9,  Spirillum  undula;  10,  Cladothrix  dichotoma.  (After 
Biitschli  from  Guilliermond  review,  Bull.  Inst.  Past.) 

FIG.  83.  —  Micros  pira  comma.  Monotrichous  bacteria.  (After  Migula  from 
Schmidt  and  Weiss.) 

FIG.  84,  —  Pseudomonas  pyocyanea.  Monotrichous  bacteria.  (After  Migula  from 
Schmidt  and  Weiss.) 

they  cannot  usually  be  seen  without  staining,  and  they  can  only  be 
stained  by  precipitating  some  chemical  which  may  add  considerably  to 
their  width.  They  are  frequently  longer  than  the  organism  which 


mi 


FIG.  85.  FIG.  86.  •  FIG.  87. 

FIG.  85. — Pseudomonas  syncyanea.  Lophotrichous  bacteria.  (After  Migula  from 
Schmidt  and  Weiss.) 

FIG.  86. — Spirillum  rubrum.  Lophotrichous  bacteria.  (After  Migula  from 
Schmidt  and  Weiss.) 

FIG.  87. — Bacillus  typhosus.  Peritrichous  bacteria.  (After  Migula  from  Schmidt 
and  Weiss,  and  Frost  and  McCampbell.) 

possesses  them  and  sometimes  many  times  that  length.  B.  sympto- 
matici  anthracis  found  in  the  soil  has  a  flagellum  sixty  times  its  own 
length.  The  arrangement  of  the  flagella  on  the  bacteria  is  quite  constant 

*  Prepared  by  W.  D.  Frost. 


106  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

and  is  used  by  some  authors  to  differentiate  genera.  Very  few  of  the 
micrococci  are  provided  with  flagella,  as  was  indicated  above,  and  in 
the  bacilli  and  spirilla  they  may  be  arranged  at  the  poles  singly  or  in 
brushes,  or  they  may  be  arranged  on  the  entire  periphery  of  the  cells. 
When  bacteria  are  provided  with  a  single  flagellum  at  one  pole,  the 
arrangement  is  said  to  be  monotrichous  (Figs.  82,  83  and  84).  When  they 
are  arranged  in  brushes,  the  arrangement  is  spoken  of  as  lophotrichous 
(Figs.  85  and  86)  and  when  they  are  arranged  on  the  entire  periphery, 
the  arrangement  is  said  to  be  peritrichous  (Fig.  87).  It  frequently 
happens  that  in  the  case  of  the  monotrichous  and  lophotrichous  the 
flagella  occur  at  both  ends  of  the  organism.  This  is  explained  by  the 
fact  that  the  organism  is  just  undergoing  binary  fission  and  that  the 
second  group  is  on  the  newly  forming  cell.  It  is  worth  while  in  this 
connection  to  call  attention  to  the  fact  that  the  flagella  on  one  end  are 
new,  while  those  on  the  other  end  may  be  thousands  of  generations  old. 

Minute  Consideration  of  Flagella.* — The  question  of  the  cilia  or 
flagella  of  bacteria  is  not  yet  entirely  decided.  The  absence  of  cilia 
in  large  bacteria  capable  of  motion  gives  the  idea  that  these  are  not  the 
only  organs  of  motion,  and  that  contraction  of  the  protoplasm  certainly 
plays  the  most  important  role  in  the  phenomena  of  motility.  More- 
over, the  nature  of  cilia  has  been  debated.  Van  Tieghem  and  Biitschli, 
taking  their  stand  primarily  on  the  difficulty  of  staining  cilia  by  the 
reagents  which  rapidly  color  protoplasm,  have  considered  these  cilia 
to  be  simply  prolongations  of  the  membrane,  lacking  all  contractibility 
and  locomotive  power.  According  to  Van  Tieghem,  when  two  cells 
formed  by  the  division  of  the  same  element  separate,  the  common  por- 
tion of  the  transverse  septum,  instead  of  dividing  neatly  in  two,  can 
stretch  out  into  a  filament  which  breaks  at  a  greater  or  less  distance  from 
each  of  the  two  daughter  cells.  This  prolongation  composes  the 
vibratile  cilium. 

This  theory,  however,  does  not  explain  the  existence  in  certain 
bacteria  of  clusters  of  cilia  at  the  two  poles,  or  of  cilia  distributed  over 
the  whole  surface  of  the  membrane.  Other  authors,  as  for  example 
A.  Fischer,  consider  the  cilia  true  prolongations  of  the  protoplasm 
issuing  through  tiny  apertures  in  the  membrane.  This  view  at  present 
tends  more  and  more  to  predominate,  and  the  existence  of  flagella  on 
bacteria  appears  to  be  demonstrated. 

•Prepared  by  A.  Guilliermond. 


BACTERIA  IO7 

Another  interesting  peculiarity,  moreover,  has  recently  been  estab- 
lished independently  by  Swellengrebel  and  by  Dangeard.  According 
to  these  authorities,  in  some  species  (Chromatium  okenii  and  Spirillum 
wlutans)  the  cilia  have  connection  with  one  of  the  chromatic  grains  of 
the  diffuse  nucleus.  There  is  a  chromatic  filament  starting  from  the 
base  of  the  cilium  and  ending  in  connection  with  a  chromatic  grain, 
similar  to  the  organisms  with  flagella  in  which  the  flagellum  is  in 
relation  to  a  basal  chromatic  grain  (blepharoplast). 

THE  HIGHER  BACTERIA* 

The  so-called  higher  bacteria  include  some  of  the  spiral  forms,  at 
least  the  larger  spirochaetes,  the  thread  or  trichobacteria,  and  the 
sulphur  or  thiobacteria. 

The  spirochaetes  and  trichobacteria  contain  so  many  forms  of 
interest  that  their  form  and  structure  needs  special  consideration. 

THE  LARGER  SPIROCH^TES. — Spirochaetes  differ  so  much  among 
themselves  that  it  seems  necessary  to  divide  them  into  two  groups. 
The  members  of  one  of  these  groups,  the  small  spirochaetes,  are  prac- 
tically identical  with  the  true  bacteria,  and  naturally  fall  in  the  family  of 
the  Spirilliacea.  Members  of  this  group,  however,  so  gradually  approach 
the  other  group,  the  large  spirochaetes,  that  it  is  difficult  to  draw  a  line 
of  separation  between  the  two,  yet  the  large  spirochaetes  resemble  in 
so  many  essential  details  the  trypanosomes  that  they  are  usually  placed 
as  a  coordinate  genus  with  them  under  the  flagellates — a  sub-class  of 
the  Protozoa.  The  larger  spirochaetes  are  described  as  follows: 

Form  and  Size. — In  form  the  spirochaetes  are  long,  very  thin  and 
flexible  spirals.  Their  length  is  usually  not  less  than  twenty  times  their 
breadth.  Some  forms  are  as  long  as  500  /*.  It  seems  probable  that 
some  of  them  are  flattened  and  hence  in  form  are  more  like  a  spirally 
bent  ribbon  than  rod. 

Motility. — These  organisms  move  very  rapidly  under  normal  con- 
ditions. The  character  of  the  movement  may  be  of  three  kinds: 
(i)  Lashing,  eel  or  snake  like;  (2)  undulatory,  compared  to  the  flapping 
of  a  sail  in  the  wind;  (3)  rotation,  similar  to  a  cork-screw  when  pushed 
into  a  cork. 

Reproduction. — Multiplication  is  by  means  of  binary  fission.  If 
these"  forms  are  to  be  considered  as  bacteria,  the  division  would  be 
expected  to  be  by  means  of  transverse  partition  walls.  A  number  of 

•  Prepared  by  W.  D.  Frost. 


IO8  MORPHOLOGY  AND    CULTURE    OF   MICROORGANISMS 

workers,  however,  have  described  a  process  of  longitudinal  division. 
Forked  forms  also  which  are  frequently  seen  are  held  to  indicate  longi- 
tudinal divisions.  Some  observers  have  claimed  that  conjugation 
occurs  among  the  spirochaetes.  If  this  is  true  their  relation  to  the 
Protozoa  would  be  quite  likely,  but  accounts  of  this  phenomenon  are 
inconclusive.  Several  observers  have  described  "rolled  up "  specimens, 
oval  and  ovoid  forms,  which  have  been  assumed  to  be  cysts.  The 
spirochaetes  break  up  into  granules  or  short  segments  and  such  speci- 
mens are  sometimes  spoken  of  as  "monili  form."  It  is  not  definitely 
known  whether  these  coccoid  forms  are  simply  degenerative  forms  or 
the  equivalent  of  bacterial  spores. 

Sheaths. — A  definite  sheath  has  been  described  for  some  forms 
and  the  irregularity  in  the  disposition  of  this  around  the  cell  may 
account  for  the  structures  that  have  been  taken  for  undulating 
membranes. 

Cell  Aggregates. — There  is  apparently  no  definite  cell  grouping  but 
tangled  masses  of  these  organisms  have  been  described  in  several 
species. 

THE  TRICHOBACTERIA. — The  trichobacteria  (Chlamydobacteriacece) 
are  thread  or  filamentous  forms.  The  cells  are  cylindrical  and  similar 
in  form  and  may  or  may  not  vary  in  size  in  different  parts  of  the  fila- 
ment. The  individual  cells  are  capable  of  independent  existence,  but 
when  growing  in  the  filament  give  evidence  of  differentiation  in  func- 
tion. Sometimes  these  filaments  are  attached  to  the  substratum  or 
some  object  in  it;  at  other  tunes  they  are  free.  In  case  of  the  sessile 
forms  the  cells  at  the  attached  end  (base)  are  smaller  than  those  at  the 
apex.  In  other  members  of  the  group  the  ends  of  the  thread  are  swollen 
or  become  club-shaped  (Fig.  88).  In  some  forms  cell  division  takes 
place. in  three  directions  of  space,  thus  forming  a  thread  of  massed  cells. 
Branching. — The  filaments  are  usually  unbranched,  but  some 
forms  show  true  branching,  such  as  is  found  among  the  plants — fungi 
and  algae.  Some  again  exhibit  what  is  called  false  branching.  This 
is  due  to  a  misplaced  cell,  which  grows  parallel  or  at  an  angle  to  the 
parent  thread  and  suggests  branching. 

Reproduction. — The  -cells  throughout  the  filament  may  divide  to 
form  spores,  but  the  apical  cells  of  the  thread  are  frequently  set  apart 
for  the  purpose  of  reproduction,  and  by  a  process  of  division  form 
spores  or  conidia.  The  conidia  are  usually  round  and  without  any 


BACTERIA 


IOQ 


resting  stage  may  produce  new  threads  of  cells.  Sometimes  spores 
germinate  while  still  in  the  old  thread  (Fig.  88),  giving  a  tangled 
mass  of  cells  or  whorls  of  new  threads  at  intervals  on  the  old.  The 
conidia  may  be  either  motile  or  non-motile.  The  motility  of  these 
conidia  when  it  exists  is  due  to  flagella. 

Sheath. — The  threads  of  cells  are  sometimes  surrounded  by  sheaths 
of  varying  thickness.     This  sheath  is  a  thickened  and  hardened  mem- 


FIG.  88 — Crenothrix  polyspora  Cohn,  Brunnenfaden.     (After  Migula  from  Schmidt 

and  Weiss.) 

brane,  and  forms  a  tube  in  which  the  different  cells  of  the  bacteria  are 
contained.  This  sheath  is  homologous  to  a  capsule.  In  it  are  fre- 
quently deposited  characteristic  by-products  of  the  cell.  In  Creno- 
thrix (an  iron  bacterium),  for  example,  we  have  iron  oxides. 

Among  the  iron  bacteria  are  several  interesting  forms.  Crenothrix 
polyspora  is  one  of  the  best  known.  Its  general  morphology  is  shown 
in  Fig.  88.  The  attached,  sessile,  threads  are  shown  at  a.  The 
tufts  of  short  threads,  radiating  from  the  larger  threads,  are 


no 


MORPHOLOGY  AND   CULTURE    OF  MICROORGANISMS 


formed  by  the  germination  of  conidia  while  they  are  still  in  the  parent 
threads.  The  large  threads,  b,  c,  d,  and  e,  show  more  details.  In  e  a 
uniform  thread  is  shown  with  the  separate  vegetative  cells;  in  d  these 
have  broken  up  into  conidia.  The  flaring  form  of  the  threads  are  shown 
in  c  and  b  where  the  conidia  are  formed  in  large  numbers.  These 
figures  also  show  the  sheath  which  is  indicated  by  the  double  line  in  c 
and  by  the  extension  of  the  lines  -beyond  the  cell  contents. 

Chlamydothrix  ochracea  Migula  is  composed  of  filamentous,  cylindri- 
cal, colorless  threads.  The  sheath  is  at  first  thin  and  colorless  but  later 
becomes  thicker,  yellow  or  brown  due  to  encrustations  of  iron  oxide. 
Multiplication  is  by  means  of  cell  division  and  swarm  cells.  These 
latter  may  sometimes  germinate  in  the  sheath,  giving  the  appearance  of 
branching  (Fig.  89,  c). 


FIG.    89. — A,  Spirophyllum  ferrugineum;   B,  Gallionella  ferruginea;    C,  Leptothrix 
ochracea.     X  about  1080.     (After  Harder.) 

Gallionella  ferruginea  Ehr.,  in  its  typical  form,  consists  of  spiral 
threads  coiled  together  in  double  or  quadruple  coils  like  a  rope.  The 
threads  are  cylindrical  but  comparatively  thin.  Individual  cells  have 
not  been  distinguished  in  the  threads  (Fig.  89,  B}. 

Spirophyllum  ferrugineum  Ellis  is  very  similar  to  and  associated 
with  the  above.  It  differs  principally  in  the  shape  of  the  threads 
which  are  flat  or  ribbon-like.  The  threads  are  always  twisted  but  may 
occur  singly ^or  be  coiled  into  ropes  (Fig.  89,  A). 


BACTERIA  III 

All  of  these  iron  bacteria  have  the  power  of  changing  certain 
soluble  salts  of  iron  into  insoluble  forms  and  thus  precipitate  them  from 
solution.  Growing  in  the  pipes  of  a  city  water  supply  their  deposits 
choke  up  the  pipes  and  hence  they  are  frequently  referred  to  as  "water 
pests."  As  a  result  of  researches  in  recent  years  these  iron  bacteria  are 
now  regarded  as  important  geological  agents  and  to  them  is  ascribed  a 
large  share  in  the  deposition  of  iron  ores. 

Other  thread  bacteria  of  considerable  importance  are  the  acti- 
nomycetacea.  Some  of  them  are  common  in  the  soil  and  recently 
have  been  given  special  study.  Others  cause  disease  and  a  well  known 
form,  Actinomyces  boms  Hartz,  is  the  cause  of  lumpy  jaw  in  cattle. 

The  actinomycetes  are  mold-like  organisms  and  often  show  true 

branching.     They  reproduce  vegetatively  or  by  means   of  conidia. 

They  are  without  sulphur  granules,  not  colored  with  bacteriopurpurin 

and  the  sheaths,  if  present,  are  not  impregnated  with  iron.     The  struc- 

I  ture  of  Actinomyces  boms  is  shown  in  Fig.  165,  p.  780,  while  the  charac- 

|  teristic  radiating  clubbed  ends  of  the  filaments,  as  these  organisms  grow 

in  the  tissues  of  cattle,  are  shown  in  Fig.  164,  p.  779. 

THE  SULPHUR  BACTERIA. — The  sulphur  bacteria  are  filamentous 
forms  which  may  reach  a  length  of  many  microns.  They  are  cylin- 
drical or  perhaps  sometimes  flat.  They  may  be  either  attached  or 
actively  motile.  The  movement  when  present  is  due  not  to  flagella, 
f  but  to  an  undulatory  motion  like  that  of  the  spirochaetes  or  Oscillaria 
among  the  algae.  As  they  move  forward  they  rotate  on  their  own  axis 
and  swing  their  free  ends. 

Spore  formation  is  unknown  in  some  forms  where  multiplication  is 
accomplished  by  the  breaking  up  of  the  threads  in  short  segments. 
In  the  case  of  the  sessile  forms  conidia  are  produced  at  the  end  of  the 
thread  and  are  motile  (Thiothrix  nivea).  The  sulphur  bacteria  contain 
at  certain  stages  strongly  refractile  sulphur  granules  in  their  bodies. 

CLASSIFICATION* 

The  classification  of  bacteria  was  early  recognized  by  Mueller  as  a 
matter  of  difficulty,  since  he  says:  "The  difficulties  that  beset  the  in- 
vestigation of  these  microscopic  animals  are  complex;  the  sure  and 
definite  determination  (of  species)  requires  so  much  time,  so  much  of 
acumen  of  eye  and  judgment,  so  much  of  perseverance  and  patience, 
that  there  is  hardly  anything  else  so  difficult." 

•  Prepared  by  W.  D.  Frost. 


112  MORPHOLOGY   AND   CULTURE    OF  MICROORGANISMS 

A  considerable  number  of  systems  for  the  classification  of  the  bac- 
teria have  been  proposed.  One  of  the  most  widely  used  at  the  present 
time  is  that  devised  by  Migula.  His  system  is  based  on  the  principle, 
universally  followed  by  botanists  and  zoologists,  of  using  morphological 
characters  only  to  distinguish  genera.  There  has  been,  however,  a 
growing  conviction  among  bacteriologists  that  it  is  necessary  to  take 
physiological  characters  into  consideration  in  determining  even  the 
major  groups  of  bacteria  in  any  system  of  classification.  This  revolu- 
tionary doctrine  was  presented  in  an  extreme  form  by  Orla  Jensen  who 
used  the  metabolic  processes  of  the  bacteria  as  the  chief  criteria  for 
establishing  not  only  genera  but  families  and  orders  as  well.  A 
Committee  of  the  Society  of  American  Bacteriologists  have  recently 
reported  on  the  Families  and  Genera  of  Bacteria*.  This  system  makes 
use  of  both  morphological  and  physiological  characters  and  promises  to 
be  an  important  step  towards  a  natural  system  of  classification.  Mi- 
gula's  system  and  that  of  the  Committee  of  the  Society  of  American 
Bacteriologists,  in  skeleton  form,  follow: 

MIGULA'S  CLASSIFICATION 

ORDERS  OF  THE  SCHIZOMYCETES 
Cells  contain  sulphur.     Colorless  or  pigmented  rose, 

violet  or  red  by  bacteriopurpurin — never  green.. THIOBACTERIA 
Cells    free    from    sulphur    and    bacteriopurpurin, 

colorless  or  faintly  colored EUBACTERIA 

FAMILIES  OF  EUBACTERIA 
Cells  globose  in  a  free  state,  not  elongating  in  any 

direction  before  division  into  i,  2  or  3  planes COCCACE^E 

Cells  cylindrical,  longer  or  shorter,  and  only  divid- 
ing in  one  plane,  and  elongating  to  twice  the 
normal  length  before  division 

1.  Cells   straight,    rod-shaped,    without  sheath, 

non-motile  or  motile  by  means  of  flagella . .  .  B  ACTERIACE^E 

2.  Cells  crooked,  without  sheath SPIRILLACE^E 

3.-  Cells  inclosed  in  a  sheath CHLAMYDOBACTERIACE^ 

GENERA  OF  THE  COCCACE^: 
Cells  without  organs  of  locomotion 

1.  Division  in  one  plane Streptococcus 

2.  Division  in  two  planes Micrococcus 

3.  Division  in  three  planes Sarcina 

Cells  with  organs  of  locomotion 

1.  Division  in  two  planes Planococcus 

2.  Division  in  three  planes Planosarcina 

*Jour.  Bact.  II,  p.  505,  1917. 


BACTERIA  113 

GENERA  OF  THE  BACTERIACE.E 

Cells  without  organs  of  locomotion Bacterium 

Cells  with  organs  of  locoomtion 

1.  Flagella  distributed  over  the  whole  body Bacillus 

2.  Flagella  polar Pseudomonas 

GENERA  OF  THE  SPIRILLACE.E 

Cells  rigid  not  snakelike  or  flexuous 

1.  Cells  without  organs  of  locomotion Spirosoma 

2.  Cells  with  organs  of  locomotion 

(a)  With  one,  very  rarely  two  or  three  polar 

flagella Microspira 

(6)   Cells  with  polar  flagella  in  tufts  of  five 

to  twenty Spirillum 

Cells  flexuous Spirochasta 

GENERA  OF  THE  CHLAMYDOBACTERIACE.E 

Cell  contents  without  granules  of  sulphur 
j .  Cell  threads  unbranched 

(a)  Cell  division  always  only  in  one  plane. .  Chlamydothrix 
(6)  Cell  division  in  three  planes  previous  to 

conidia  formation 

i.  Cells     surrounded     by     a     very 
delicate,  scarcely  visible,  sheath 

"(marine) Phragmidiothrix 

ii.  Sheath   clearly   visible    (in   fresh 

water) Crenothrix 

2.  Cell  threads  branched  (pseudobranches) Sphaerothrix 

FAMILIES  OF  THE  THIOBACTERTA 

Filamentous  bacteria  which  do  not  contain  bac- 

teriopurpurin.  Cells  contain  sulphur  granules . .  BEGGIATOACE^E 

Cells  contain  bacteriopurpurin,  sulphur  granules 

may  also  be  included RHODOBACTERIACE^ 

GENERA  OF  THE  BEGGIATOACE./E 

Cells  non-motile,  threads  attached  to  some  object.  .Thiothrix 
Moves  by  means  of  an  undulating  membrane Beggiatoa 

GENERA  OF  THE  RHODOBACTERIACE.E 

This  family  includes  twelve  genera  as  follows:  Thiocystis,  Thiocapsa,  Thiosarcina, 
Lamprocystis,  Thiopedia,  Amcebobacter,  Thiothece,  Thiodictyon,  Thiopoly- 
coccus,  Chromatium,  Rhodochromatium  and  Thiospirillum. 


114  MORPHOLOGY  AND   CULTURE   OF   MICROORGANISMS 

THE  FAMILIES  AND  GENERA  OF  THE  BACTERIA 

Report  of  the  Committee  of  the  Society  of  American  Bacteriologists.     C.-E.  A. 
Winslow  et  al.     (Artificial  key) 

ORDERS  OF  THE  SCHIZOMYCETES 

Cells  united  during   the   vegetative  stage  into   a 

pseudoplasmodium MYXOBACTERIALES 

Cells  not  forming  a  pseudoplasmodium 

Cells  free  or  united  in  elongated  filaments,  often 
with   a   well   defined   sheath.     Conidia  fre- 
quently   formed.     Free    sulphur,     iron    or 
bacteriopurpurin  often  present. 
Cells  typically  containing  granules  of  sulphur  or 

bacteriopurpurin  or  both THIOBACTERIALES 

Sidphur  and  bacteriopurpurin  absent;  iron  often 

present CHLAMYDOBACTERIALES 

Cells  necer  in  sheathed  filaments.  Conidia  only 
in  mycelial  Mycobacteriaceae.  Flagella  often 
present.  Free  iron,  sulphur,  or  bactiopurpurin 
never  present. EUBACTERIALES 

FAMILIES  OF  THE  EUBACTERIALES 

Cells  spiral  with  polar  flagella IV.  SPIRJLLACE^E 

Not  as  above 

Cells  spherical;  rarely,  if  ever,  motile;  spores 
never  produced;  never  securing  growth  energy 

from  nitrogen  or  ammonia V.  COCCACE^ 

Not  as  above 

Cells  short  rod-shaped  with  a  single,  rarely  two, 
polar   flagellum;    usually   forming  green   or 

yellow  pigment III.  PSEUDOMONADACE^E 

Not  wholly  as  above 

Spores  formed VIII.  BACILLACE/E 

Spores  never  formed 

Metabolism  simple,  securing  growth  energy 
from  carbon,  hydrogen,  or  their  simple 

compounds;  flagella,  if  present,  polar I.  NITROBACTERIACE^: 

Metabolism  complex,  dependent  upon  more 
complex  carbohydrate  and  protein  sub- 
stances; flagella,  if  present,  peritrichic. 
Cells  clubbed,  fusiform,  filamentous, 
branching  or  mycelial;  those  not  distinctly 
so  are  either  acid-fast  or  show  barred 

irregular  staining IT.  MYCOBACTERIACE^ 

Not  as  above 

Gram  positive;  non-motile VI.  LACTOBACILLACE^ 

Gram  negative;  often  motile VI.  BACTERIACE^: 


BACTERIA  115 

GENERA  OF  THE  EUBACTERIALES 

I.  NITROBACTERIACE^E 

Fixing  nitrogen  or  oxidizing  its  compounds 
Fixing  free  nitrogen 

Cells  large;  in  soil 7.  Azotobacter 

Rods  minute;  in  roots  of  leguminous 

plants 8.  RMzobium 

Oxidizing  nitrogen  compounds 

Oxidizing  ammonia 5.  Nitrosomonas 

Oxidizing  nitrites 6.  Nitrobacter 

Not  as  above 

Oxidizing  hydrogen i.  Hydrogenomonas 

Oxidizing  carbon  compounds 
Oxidizing    alcohol;    branching    forms 

common 4.  Mycoderma 

Not  as  above,   using  simpler  carbon 
compounds 

Oxidizing  CO 3.  Carboxydomonas 

Oxidizing  CH4 2.  Methanomonas 

II.  MYCOBACTERIACE^: 

Slender  rods  staining  with  difficulty  and 

acid  fast 3.  Mycobacterium 

Not  as  above 

Mycelium  and  conidia  formed 
With  aerial  hyphae  and  conidia;  usually 

saprophytic  soil  organisms 2.  Nocardia 

Hyphae  and  conidia  not  aerial;  usually 

parasitic  in  animals i.  Actinomyces 

Not  as  above;  cells  rod-like,  usually  somewhat 
curved,  clubbed,  fusiform,  or  even 
branched,  but  never  mycelial 
Thick,  long  threads,  fragmenting  into 

short  thick  rods 6.  Leptotrichia 

Not  as  above 

Cells  usually  elongate  and  fusiform, 
filaments,  if  formed  not  branch- 
ing; stains  somewhat)  irregularly.  .5.  Kusiformis 
Cells  slightly  curved,  clubbed,  or  in 
old  cultures  even  branching;  not 
filamentous;  showing  definite  bar- 
red staining 4-  Corynebacterium 

III.  PSEUDOMONADACE.E 

Generic  characters  mainly  those  of  family.  .1.  Pseudomonas 


Il6  MORPHOLOGY  AND    CULTURE    OF   MICROORGANISMS 

IV.  SPIRILLACE^: 

Flagellum  single  (rarely  2  or  3) i.  Vibrio 

Flagella  tufted  (5  to  20) 2.  Spirillum 

V.  COCCACE^E 

Abundant  red-pigmented  growth  on  agar.  .  7.  Rhodococcus 
Not  as  above 
Gram  negative 

Normally  in   pairs  of  flattened  cells; 
growth  on  plain,  agar  scanty,  never 

bright  yellow i.  Neisseria 

Normally  in  plates,  packets,  or  irregu- 
lar masses;  growth  on  plain  agar 
abundant,  pigment  definitely 
yellow 

Cells  in  regular  packets 6.  Sarcina 

Cells  not  in  regular  packets 5.  Micrococcus 

Gram  positive  (exceptions  rare  and  not 

easily  confused  with  above  genera) 
Cells  normally  in  chains,  sometimes  in 
pairs  (especially  in  acid  environment) 
never  in  large  irregular  masses. 
Gelatin  rarely  liquefied.  Growth  on 
plain  agar  usually  translucent,  never 

heavy,  never  yellow  or  orange 2.  Streptococcus 

Cells  normally  in  groups  and  masses; 
(occasionally  in  plates  in  Albo- 
coccus)  chains  short  and  irregular, 
if  present.  Gelatin  often  lique- 
fied. Agar  growth  abundant, 

white  to  orange 

Pigment    orange    (rarely    lacking); 

gelatin  often  liquefied  actively.. .  .3.  Staphylococcus 
Whitish  to  porcelain  white;  liquefac- 
tion less  vigorous 4.  Albococcus 

VI.  BACTERIACE.E 

Plant  pathogens 2.  Erwinia 

Not  as  above;   saprophytes  or  in  animal 

habitats  (intestines,  tissues,  etc.) 
Usually  motile  and  exhibiting  active 
fermentative  powers;  typically  para- 
sitic in  intestines  of  man  and  higher 
animals;  growing  well  on  ordinary 
media .  .1.  Bacterium 


BACTERIA  117 

Not  wholly  as  above 

Growing  only  in  presence  of  hemo- 
globin, ascitic  fluid  or  serum 4.  Hemophilus 

Growth  on  media  scanty,  but  less 
sensitive  than  the  above;  short  rods 
with  tendency  to  bipolar  stain 3.  Pasteurella 

VII.*LACTOBACILLACE,E 

Generic  characters  mainly  those  of  family.  .  i.  Lactobacillus 

VIII.  BACILLACE^E 

Aerobic,    usually    saprophytic;    cells    not 

greatly  enlarged  (if  at  all)  at  sporulation.  i.  Bacillus 
Anaerobic,    often    saprophytic;    cells    fre- 
quently enlarged  at  sporulation 2.  Clostridium 

NOMENCLATURE 

It  is  most  important  that  each  kind  of  bacterium  should  have  a 
definite  name.  The  name  should  be  a  binomial  and  not  a  trinomial. 
It  is  also  very  desirable  that  all  bacteriologists  should  adhere  to  the  rules 
that  govern  botanists  in  these  matters.  Probably  the  most  important 
points  to  remember  are:  To  use  Latin  names  for  all  groups;  to  recognize 
only  one  valid  designation  for  each  organism  or  group  and  that  the 
oldest  (with  certain  limitations);  to  designate  orders  with  the  ending 
ales,  families  with  the  ending  aceae,  sub-families  with  oideae,  tribes  with 
eae,  and  sub-tribes  with  inae-,  to  use  generic  names  as  substantives  and 
write  them  with  a  capital  letter;  to  designate  all  species  by  the  name  of 
the  genus  and  a  specific  name  or  epithet,  usually  of  the  nature  of  an 
adjective  (the  two  names  forming  a  binomial  or  binary  name). 

RELATIONSHIP  OF  BACTERIA* 

There  has  been  a  great  deal  of  discussion  as  to  whether  bacteria 
are  plants  or  animals.  They  were  first  described  as  animalcula  and 
to  the  popular  mind  they  are  usually  animals  or  "bugs."  It  is  dim- 
cult  to  determine  their  exact  relation  philogenetically.  These  diffi- 
culties are  so  great  that  some  scientists,  as  Haeckel,  would  create  a 
new  kingdom,  call  it  Protista,  and  put  in  it  some  of  the  lower  plants 
and  animals  which  are  difficult  to  classify,  together  with  the  bacteria. 
The  bacteria  are  undoubtedly  more  closely  related  to  the  blue-green  algae 
than  to  any  other  forms  of  life.  They  resemble  these  organisms  in  form, 
method  of  reproduction,  and  absence  of  definite  nucleus.  It  is  quite 

•  Prepared  by  W.  D.  Frost. 


Il8  MORPHOLOGY  AND    CULTURE   OF   MICROORGANISMS 

impossible  to  decide,  furthermore,  whether  some  forms,  such  as  Bact.  viride 
and  Bact.  chlorinum,  are  blue-green  algae  or  bacteria.  On  the  other 
hand,  there  are  some  points  of  resemblance  between  the  bacteria  and 
the  protozoa.  Spore  formation,  similar  to  that  among  the  bacteria, 
occurs  among  some  of  the  protozoa.  Another  point  of  resemblance  is 
the  possession  of  flagella.  Some  of  the  flagellates  quite  closely  resemble 
the  bacteria  in  many  ways,  and  the  Spirochafat  which  are  usually 
believed  to  be  bacteria,  have  been  classed  as  flagellates  by  eminent 
protozoologists. 

Physiologically  the  bacteria  are  quite  closely  related  to  the  fungi, 
and  are  frequently  classed  with  them  under  the  term  Schizomycetes. 

ARTIFICIAL  CULTIVATION  OF  BACTERIA* 

The  introduction  of  methods  of  artificial  cultivation  marks  the  beginning  of  the 
science  of  microbiology.  These  methods  were  developed  by  Pasteur  and  Koch  and 
are  depended  upon  by  the  microbiologist  of  to-day  as  the  foundation  for  most  of  his 
work.  It  has  been  the  aim  of  investigation  to  discover  a  more  general  culture 
medium.  So  far  it  has  been  impossible  to  do  this,  but  beef  broth,  made  after  a 
formula  suggested  by  LoefHer  many  years  ago,  forms  the  basis  of  nearly  all  of  our 
culture  media.  This  beef  broth,  or  nutrient  bouillon,  is  made  by  extracting  meat 
free  from  fat  in  water,  adding  a  small  per  cent  of  peptone,  correcting  the  chemical 
reaction,  clarifying  and  sterilizing.  To  this  broth  various  substances  are  added 
for  special  purposes;  gelatin  and  agar,  in  order  to  solidify  the  media,  and  various 
sugars  and  other  chemical  substances  for  the  purpose  of  determining  the  physiological 
characteristics  of  various  bacteria.  One  of  the  difficulties  with  the  present  methods 
of  the  artificial  cultivation  of  bacteria  is  the  inconstancy  of  the  composition  of  the 
media,  due  to  the  fact  that  the  extract  of  beef,  the  peptone,  and  other  ingredients, 
cannot  be  obtained  chemically  pure.  If  it  should  prove  possible  to  use  synthetic 
substances,  such  as  the  polypeptids,  it  would  mark  a  great  step  in  advance,  but  it  is 
probably  quite  impossible  to  devise  a  single  medium  upon  which  all  bacteria  will 
grow.  Some  bacteria,  such  as  those  which  produce  nitrification,  refuse  to  grow  on 
ordinary  media  containing  organic  material.  The  cultivation  of  bacteria  in  pure 
culture  is  dependent  upon  isolation,  and  the  method  of  isolation  suggested  by  Robert 
Koch  in  1880,  and  known  as  the  plate  culture  method,  has  given  eminent  satis- 
faction. This  method  is  dependent  upon  the  use  of  liquefiable  solid  media,  such 
as  gelatin  or  agar. 

*  Prepared  by  W.  D.  Frost. 


CHAPTER  V 
FILTRABLE  MICROORGANISMS* 

The  terms  "filtrable  microorganisms"  and  "filtrable  viruses" 
are  used  to  designate  a  group  of  disease-producing  microorganisms  that 
are  characterized  by  their  ability  to  pass  through  ordinary  "  bacteria- 
proof  "  niters.  In  the  past  it  has  been  customary  to  speak  of  these  filter 
passers  as  invisible  or  ultramicroscopic  because  of  the  fact  that,  besides 
being  filtrable,  they  were,  with  the  single  exception  of  the  virus  of 
bovine  pleuro-pneumonia,  invisible  under  the  microscope  and  incapable 
of  multiplying  in  vitro  on  any  of  the  usual  culture  media.  Recent 
discoveries  indicate  that  the  terms  "invisible"  and  "ultramicroscopic" 
are  incorrect,  at  least  with  respect  to  some  members  of  the  group. 
So  long  as  clear,  filtered  fluids  that  gave  no  visible  evidence  of  life  were 
capable  of  setting  up  infectious  disease  in  men  or  in  animals  there  was 
some  reason  for  the  use  of  those  terms  and  even  for  Beijerinck's  fanciful 
conception  of  a  "living  fluid  contagion."  However,  the  brilliant 
researches  of  Noguchi  have  offered  a  technique  by  means  of  which  some 
of  these  hitherto  invisible  viruses  have  been  cultivated  outside  of  the 
animal  body  and  made  visible  under  the  microscope.  While  some 
members  of  this  group  may  indeed  be  of  ultramicroscopic  size,  there  is 
reason  to  believe  that  many  of  them  will  eventually  be  rendered  visible 
through  improvements  in  bacteriological  technique. 

The  characteristics  of  the  filtrable  viruses  may  be  best  understood 
by  consideration  of  a  typical  example,  the  virus  of  foot-and-mouth 
disease.  In  this  disease  vesicles  form  in  the  mouths  and  on  the  feet 
of  infected  cattle.  The  virus  is  known  to  be  present  in  the  lymph 
which  forms  in  these  vesicles  because  this  lymph  will  produce  typical 
attacks  of  foot-and-mouth  disease  when  inoculated  into  susceptible 
animals.  If  now  this  infectious  lymph  be  diluted  with  water  and  passed 
through  a  Berkefeld  filter  the  resulting  filtrate  will  be  found  to  be  free 
from  all  visible  microorganisms  and  in  addition  the  usual  culture  tests 
will  give  negative  results.  Notwithstanding  this  apparent  sterility, 

•  Prepared  by  M.  Dorset. 

119 


120 


MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 


FIG.  90. — Apparatus  for  fractional  filtration,  designed  for  use  with  Pasteur- 
Chamberland  or  Berkefeld  niters,  a,  Glass  mantle  surrounding  filter;  b,  Chamber- 
land  filter;  c,  paraffin  joint;  d  and  e,  rubber  stoppers;/,  double  side-arm  suction  flask; 
g,  pinchcock  controlling  outlet  from  suction  flask;  h,  outlet  tube  surrounded  by  glass 
shield  and  attached  to  lower  end  of  suction  flask  by  means  of  short  rubber  tubing; 
i,  glass  shield  fused  to  and  surrounding  outlet  tube  as  a  protection  against  contamina- 
tion when  the  nitrates  are  drawn  off;  j,  glass  inlet  tube  plugged  with  cotton,  for  ad- 
mitting air  into  suction  flask;  k,  pinchcock  governing  the  admission  of  air  into  flask; 
I,  vacuum  gauge;  m,  stopcock  connected  with  vacuum  pump.  (U.  S.  Dept.  of  Agri- 
culture, Bureau  of  Animal  Industry,  Butt.  113.) 


FILTRABLE  MICROORGANISMS  121 

however,  the  filtrate  will  produce  disease  in  cattle  in  the  same  manner 
as  the  unfiltered  lymph.  It  is  known  that  the  symptoms  produced 
by  the  filtrate  are  caused  by  a  living  organism  and  not  by  a  toxin, 
because  by  successive  nitrations  and  inoculations  the  disease  can  be 
transmitted  through  a  long  series  of  animals,  thus  indicating  clearly 
that  there  exists  in  the  filtered  lymph  a  living  organism  which  is  capable 
of  reproduction.  Another  proof  that  the  virulence  of  the  filtered  lymph 
is  caused  by  the  presence  of  living  corpuscular  elements,  and  that  it  is 
not  a  mere  solution  of  a  toxin,  is  found  in  the  failure  of  the  virus  to 
pass  through  filters  of  finer  grain  than  the  Berkefeld  as,  for  example, 
the  Kitasato  filter.  The  microorganism  of  foot-and-mouth  disease  has 
not  been  cultivated  nor  made  visible.  Among  other  diseases  produced 
by  filtrable  viruses  which  as  yet  remain  invisible,  are  hog  cholera, 
rinderpest,  swamp  fever,  fowl  plague  and  South  African  horse  sickness. 

The  invisibility  of  this  group  of  microorganisms  may  depend  upon 
either  their  minute  size  or  their  peculiar  structure.  The  most  powerful 
microscopes  will  not  enable  us  to  discern  with  distinctness  objects  which 
are  less  than  o.iju  in  diameter.  We  know  of  bacteria  which  in  size 
approach  this  limit  quite  closely  (M.  progrediens,  0.15/1  ^n  diameter) 
and  there  is  no  reason  for  believing  that  the  size  of  organisms  is  limited 
by  our  ability  to  see  them.  As  already  stated,  invisibility  may  also 
result  from  a  peculiarity  of  structure,  such  as  complete  transparency 
and  failure  to  stain  with  the  reagents  ordinarily  used  for  this  purpose. 

The  ability  of  microorganisms  to  pass  through  filters  is  dependent 
upon  a  variety  of  factors.  The  size  and  plasticity  of  the  organism, 
the  fineness  of  the  pores,  and  the  thickness  of  the  walls  of  the  filter  as 
well  as  the  conditions  under  which  the  filtration  is  performed,  will  all 
influence  the  result. 

The  failure  of  the  filtrable  microorganisms  to  develop  under  artificial 
conditions  is  to  be  attributed  to  their  strict  parasitism  and  to  our  in- 
ability to  imitate  exactly  in  the  laboratory  the  conditions  which  exist 
in  the  animal  body.  The  method  of  Noguchi,  referred  to  above,  and 
which  has  done  so  much  to  advance  our  knowledge  of  the  filtrable 
viruses,  was  first  used  to  cultivate  Treponema  pallidum.  The  culture 
medium  is  placed  in  long  narrow  test  tubes  and  consists  of  a  piece  of 
fresh  sterile  rabbit's  kidney  placed  in  the  bottom  of  the  tube  over  which 
is  poured  sterile  unheated  and  unfiltered  ascitic  fluid  or  a  mixture  of 
ascitic  fluid  and  agar.  The  surface  of  this  medium  is  covered  with  a 


122  MORPHOLOGY  AND   CULTURE   OF  MICROORGANISMS 

layer  of  sterile  paraffin  oil  to  exclude  oxygen.  The  material  from  which 
cultures  are  to  be  made  is  introduced  into  the  bottom  of  the  tube  by 
means  of  capillary  pipettes.  * 

While  the  filtrable  microorganisms  possess  certain  qualities  in  com- 
mon, in  some  respects  they  differ  widely  from  one  another.  Some  will 
pass  only  through  the  coarsest  of  bacteria-proof  filters,  while  others  pass 
readily  through  the  densest  filters,  thus  indicating  wide  differences  in 
size  or  in  structure.  Some  are  very  susceptible  to  the  action  of  germici- 
dal  agents,  whereas  others  are  more  resistant  than  the  ordinary  bacteria. 
Some  produce  disease  in  only  one  species  of  animal,  while  others  show 
little  or  no  limitation  in  this  respect.  The  diseases  produced  by  these 
microorganisms  likewise  differ  markedly,  some  being  comparatively 
benign  and  local  in  character,  whereas  others  appear  as  the  most  pro- 
found septicaemias.  Some  are  extremely  contagious,  while  others  can 
be  transferred  from  one  animal  to  another  only  by  means  of  an  inter- 
mediate host.  In  fact  these  invisible  microorganisms  seem  to  differ 
among  themselves  quite  as  widely  as  do  those  which  are  visible  to  us. 
The  existence  of  a  filtrable  microorganism  is  determined  as  follows: 
The  infectious  agent  must  pass  through  a  bacteria-proof  filter,  which 
is  free  from  imperfections  as  shown  by  tests  with  visible  organisms  of 
small  size.  Pressure  exceeding  one  atmosphere  should  not  be  employed 
during  filtration.  The  time  of  filtration  should  not  exceed  one  hour. 
The  filtrate  should  remain  free  from  all  visible  bacteria  as  shown  by 
microscopic  examination  and  cultural  tests.  The  filtrate  should 
possess  the  specific  disease-producing  qualities  of  the  unfiltered  material. 
Animals  infected  with  the  filtrate  should  yield  material  which,  after 
filtration,  will  in  its  turn  possess  the  attributes  of  the  original  unfiltered 
material.  Recent  suggestive  developments  have  thrown  some  light  on 
the  possible  nature  of  filtrable  viruses.  The  reader  is  referred  to  the 
work  of  Flexner  and  Noguchi  since  1912,  published  in  the  Journal 
of  Experimental  Medicine;  he  is  also  requested  to  read  the  article  by 
Lohms  and  Smith  already  mentioned  on  page  99. 

*  For  details  of  this  method  see  J.  Exp.  Med.,  1911,  et  seq. 


CHAPTER  VI 
PROTOZOA* 

INTRODUCTION 

Many  of  the  diseases  which  are  known  to  be  due  to  an  infecting 
agent  are  caused  by  bacteria;  but  others  are  caused  by  protozoa. 

The  bacteria  belong  to  the  vegetable  kingdom.  The  protozoa  are 
unicellular  animals;  they  are  extremely  numerous  and  are  very  widely 
distributed  in  nature.  They  occur  in  water,  soil  and  in  the  bodies  of 
most  animals. 

From  a  zoological  point  of  view,  the  protozoa  constitute  an  impor- 
tant sub-kingdom.  It  is  sometimes  difficult  to  say  whether  a  minute 
organism  is  a  plant  or  an  animal.  For  this  reason,  primitive  unicellular 
organisms  are  sometimes  classified  by  themselves,  as  Protista  (pages  1 1 , 
1 1 7) ,  a  kingdom  which  thus  includes  not  only  primitive  organisms  which 
have  not  yet  been  definitely  established  in  either  group  but  also  certain 
unicellular  animals  and  plants.  It  appears  preferable,  however,  to 
determine  as  far  as  possible  the  genetic  relationship  of  various  or- 
ganisms and,  by  the  study  of  their  physiology  and  modes  of  develop- 
ment to  differentiate  between  those  which  are  plant-like  and  those 
which  are  animal-like  in  character.  The  protozoa  are  thus  included 
in  the  animal  kingdom  and  have  been  defined  as  "unicellular  animals." 
They  are  to  be  distinguished,  on  the  one  hand  from  primitive  forms  such 
as  bacteria  which,  lacking  differentiation  of  nucleus  and  cytoplasm, 
do  not  conform  to  the  type  of  structure  of  true  cells,  and  on  the  other 
hand,  from  primitive  unicellular  organisms  of  plant-like  character  such 
as  algae  and  fungi,  f 

Many  protozoa  live  in  fresh  water.  Others  live  in  the  sea;  chalk  is 
formed  from  the  skeletons  of  myriads  of  protozoa  which  once  lived  in 
the  ocean.  While  a  large  proportion  of  the  protozoa  are  free-living, 
others  are  parasitic  on  animals  and  plants.  Some  of  the  parasitic 
protozoa  are  practically  harmless  and  do  no  apparent  injury  to  the 

•  Prepared  by  J.  L.  Todd. 
t  See  page  13. 

123 


124 


MORPHOLOGY  AND    CULTURE    OF   MICROORGANISMS 


hosts  which  support  them;  others  produce  severe  diseases.  Before 
mentioning  those  especially  which  cause  disease  (see  page  876)  it  will 
be  well  to  consider  the  protozoa  as  a  class  and  to  discuss  the  characters 
which  all  have  in  common. 

STRUCTURE  OF  THE  PROTOZOA 

Most  protozoa  are  so  small  as  to  be  visible  only  by  the  aid  of  the 
microscope  but  certain  species  are  visible  to  the  naked  eye  as  individuals, 


FIG.  91. — Amceba  vespertilio.     (After  Doflein.) 


or  as  agglomerated  masses  of  individuals.  For  example,  the  Sarco- 
sporidia,  which  occur  in  the  muscles  of  mice  and  other  animals,  can 
easily  be  seen  without  a  microscope,  and  the  huge  plasmodial  masses 
of  Mycetozoa,  which  are  sometimes  seen  on  rotting  wood  or  in  tan 
pits,  may  measure  many  centimeters  in  breadth. 

Like  all  living  things,  the  protozoa  are  composed  of  protoplasm  (page 
1 8)  and  its  products.  Protoplasm  is  a  complex  mixture  of  various  sub- 
stances in  a  colloidal  condition.  When  studied  by  appropriate  methods, 


PROTOZOA  125 

the  protoplasm  of  a  cell  appears  to  be  alveolar  or  foam-like  in  structure. 
This  is  because  the  protoplasm  is  emulsoidal  in  character  being  com- 
posed of  a  mixture  of  many  more  or  less  non-miscible  substances, 
some  of  which  are  fluid  in  character,  others  more  of  the  nature  of 
solids.  In  such  a  mixture,  the  more  viscid  materials  form  tiny 
globules,  and  each  of  these  is  surrounded  by  a  layer  of  softer  material 
(Fig.  91).  Consequently,  cytoplasm  is  alveolar  in  structure;  it  has  an 
appearance  similar  to  that  produced  by  the  myriads  of  bubbles  in  a 
mass  of  foam.  The  walls  of  the  outer  layer  of  alveoli,  or  of  alveoli 
which  surround  a  resistant  structure  within  the  cell,  are  perpendicular 
to  the  surface  against  which  they  lie  but  the  outline  of  the  alveoli, 
which  are  not  in  contact  with  a  firm  structure,  is  more  nearly  circular. 
An  exactly  similar  arrangement  of  the  alveoli  may  be  seen  in  a  mass  of 
soapsuds  contained  in  a  bottle;  wherever  the  bubbles  touch  an  un- 
yielding surface,  their  outline  becomes  rectangular. 

Recent  studies  in  colloidal  chemistry  and  in  the  microscopic  dissection 
of  cells  have  furnished  valuable  contributions  to  the  knowledge  of  the 
chemical  and  physical  properties  of  protoplasm.  The  view  has  been 
advanced  that  protoplasm  consists  largely  of  material  in  a  state  known 
in  colloidal  chemistry  as  a  gel,  some  portions  being  firm  and  viscid 
and  others  very  soft  in  character.  Procedures  which  convert  such 
material  into  a  sol  or  fluid  state  are  said  to  cause  the  protoplasm  to 
quickly  disintegrate.  Certain  portions  of  the  cell  such  as  the  limiting 
membrane,  the  nuclear  membrane  and  the  nucleolus  are  of  firmer 
consistence  than  other  portions,  and  some  cells  contain  globules  and 
granules  of  various  types. 

The  protoplasm  of  a  protozoon  may  be  divided  into  two  main 
portions:  the  cytoplasm  and  the  nucleus  (Chapter  I).  The  cytoplasm, 
as  a  whole,  may  be  divided,  more  or  less  easily,  into  a  clearer,  denser, 
more  resistant  outer  layer — the  ectoplasm;  and  a  more  fluid,  granular, 
internal  portion — the  endoplasm.  Denser,  more  resistant  fibers  some- 
times run  through  the  cytoplasm  and,  like  a  skeleton,  serve  to  fix  the 
shape  of  the  organism  in  which  they  exist. 

The  nucleus,  in  its  simplest  form,  is  a  structure  which  is  differ- 
entiated from  the  remainder  of  the  cell  by  being  more  refractile  and 
by  being  colored  more  deeply  in  specimens  which  have  been  stained 
by  dyes.  It  stains  deeply  because  it  contains  a  substance  called  chro- 
matin.  The  chromatin  usually  occurs  in  granules  which  may  vary 


126  MORPHOLOGY  AND   CULTURE    OF   MICROORGANISMS 

considerably  in  size  and  which  are  supported  upon  a  linin  framework 
that  does  not  stain  by  ordinary  methods.  The  interstices  of  the 
nucleus  are  filled  with  nuclear  sap.  A  limiting  nuclear  membrane 
may  be  present,  but  it  is  not  an  essential  part  of  the  nucleus.  The 
nuclear  material  may  be  all  gathered  together  in  a  single  mass,  or  it 
may  be  distributed  in  small  granules  termed  chromidia  so  that,  at  the 
first  glance,  no  nucleus  seems  to  be  present.  Such  chromidia  may  be 
said  to  constitute  a  distributed  nucleus,  although  the  term  nucleus  is 
usually  applied  to  a  well  differentiated  cell  structure. 

The  nucleus  (page  15)  is  to  be  regarded  as  the  most  important  unit 
in  the  structure  of  the  cell  and  is  apparently  essential  for  the  con- 
tinued existence  of  the  latter.  If  cells  are  divided  portions  contain- 
ing no  nucleus  invariably  die  while  portions  containing  the  nucleus 
may  continue  to  live  and  eventually  recover  from  the  injury.  The 
r61e  of  the  nucleus  is  not  fully  understood  but  it  seems  certain  that  it 
is  a  controlling  center  for  the  cell's  activities.  It  is  concerned  in  the 
nutrition  of  the  cell,  frequently  nuclear  structures  have  to  do  with  the 
motility  of  cells  and  the  chromatin  serves  as  a  medium  for  the 
hereditary  transmission  of  specific  characteristics.  Its  functions, 
therefore,  are  at  least  three-fold  since  it  is  active  in  trophic,  kinetic 
and  reproductive  capacities.  Usually,  all  these  functions  are  subserved 
by  a  single  nucleus;  sometimes,  however,  as  in  the  flagellates  and 
many  ciliates  they  are  divided  between  two  nuclei  (page  18). 

ACTIVITIES  OF  THE  PROTOZOA 

The  higher  animals  or  Metazoa  are  composed  of  a  great  number 
of  cells.  A  protozoon  consists  of  a  single  cell.  In  the  former  the 
various  functions  of  the  body  are  each  carried  out  by  a  special  type 
of  cell;  for  example,  movement  is  performed  by  the  muscle  cells, 
digestion  is  provided  for  by  the  cells  of  the  alimentary  tract,  and  urine 
is  excreted  by  the  kidney  cells.  A  protozoon  being  a  unicellular 
animal,  these  various  functions  must  be  performed  within  the  single 
cell  of  which  it  consists.  Consequently  certain  parts  of  its  protoplasm 
are  especially  differentiated  and  function  in  a  manner  similar 
to  the  organs  of  multicellular  animals.  Such  differentiated  parts  are 
termed  organella  and  by  means  of  these  the  protozoa  move  about, 
feed,  and  excrete  waste  products  in  many  respects  like  the  higher 
animals. 


PROTOZOA 


127 


The  activities  of  a  protozoon  may  be  considered  under  LOCOMOTION, 

METABOLISM*  and  REPRODUCTION. 

LOCOMOTION. — The  protozoa  have  several  different  modes  of  mov- 
ing themselves  about.  Some  of  them  move  by  the  formation  of 
temporary  processes  or  pseudopodia;  in 
this  method  of  progression,  the  protoplasm 
flows  out,  in  finger-like  processes,  from  the 
body  of  the  organism  and,  as  the  protoplasm 
flows  into  these  processes,  the  whole  organ- 
ism progresses,  literally,  by  flowing  along. 
Some  of  the  gregarines  move  about  by 
means  of  a  flowing  of  the  protoplasm  which 
always  takes  place  in  one  direction;  it  is 
probable  that  |the  control  of  the  direction 
of  the  flow  in  these  parasites  is  effected  by 
the  contraction  of  myonemes.  These  are 
contractile  fibers,  which  usually  lie  near  the 
surface  of  the  organism  possessing  them. 
Through  their  contraction,  the  form  of  the 
body  of  the  parasite  may  be  altered  and,  in 
this  way,  motion  may  be  produced.  Cilia 
are  small  hair-like  processes,  which  may 
occur  either  in  definite  areas  or  in  large 
numbers  over  the  whole  surface  of  a  proto- 
zoon. They  produce  motion  by  waving 
and,  acting  together,  make  a  strong  simul- 
taneous stroke  in  one  common  direction, 
ic  movement  of  all  the  cilia  of  an  organ- 

jm  is,  however,  usually  not  synchronous 
>ut  proceeds  in  waves  across  the  surface 
>f  its  body  so  that  the  appearance  is  simi- 

ir  to  that  produced  when  a  breeze  passes 

:ross  a  field  of  grain.    Flagella  are  larger 

tan   cilia;    they   are    whip-like   processes 

rhich    have    a  lashing   movement.     They 

ire  usually  few  in  number  and  are  often  placed  at  the  ends  of  the  or- 

inism.     Undulating  membranes  consist  either  of  a  thin  fold  of  the  sur- 
layer  or  of  rows  of  fused  cilia  and  form  either  fin-like  organs  ex- 

*(Seep.  195.) 


cv.'- 


FIG.  92.  — Paramecium 
caudatum:  division  showing 
the  macronucleus  (N)  divid- 
ing without  mitosis,  the  mi- 
cronucleus  (ri)  dividing  mi- 
totically.  c.z>i.,Old,  andc.v2., 
new,  contractile  vacuoles. 
(Minchin,  after  Butschli  and 
Schewiako/,  in  Leuchart 
and  Nitsche's  Zoologische 
Wandtaflen,  No.  LXV.) 


128 


MORPHOLOGY   AND    CULTURE    OF   MICROORGANISMS 


tending  along  the  surface  of  the  organisms  or  special  organs  for  the 
intake  of  food. 

REPRODUCTION 

The  protozoa  reproduce  in  many  different  ways  and  several  of  these 
ways  may  occur  in  a  single  organism.  For  this  reason,  their  repro- 
ductive power  is  very  great;  in  power  of  repeating  their  like,  they  fall 
just  short  of  the  bacteria.  The  union  of  a  male  and  a  female  form  does 


FIG.  93. — Stages  in  the  division  of  Amoeba  poly  podia. 

from  Doflein.) 


(After  F.  E.  Schulze  and  Lange 


not  always  precede  multiplication;  sexual  union  and  reproduction, 
though  now  combined  in  many  animals,  may  have  been  originally  two 
entirely  distinct  phenomena  and,  in  the  protozoa,  though  sexual  union 
may  be  concerned  with  the  production  of  new  individuals,  it  is  often 
especially  associated  with  the  regeneration  of  the  protoplasm  of  the 
parasites  taking  part  in  it. 

The  simplest  of  the  methods  of  reproduction  is  simple  binary  divi- 
sion, in  which  the  organism  divides  into  two  equal  parts.  A  modifica- 
tion of  this  process  is  gemmulation,  in  which  a  small  protozoon  buds  off 


PROTOZOA 


I2Q 


from  a  larger  parent;  sometimes  many  buds  are  formed  rapidly,  one 
after  the  other,  until  the  parent  protozoon  disappears  in  a  swarm  of 
daughter  cells.  When  a  protozoon  divides  at  a  single  division  to  pro- 
duce a  large  number  of  daughter  cells  simultaneously,  the  process  is 


FIG.  94. — Coccidium  schubergi.    A-C,  asexual  multiplication;  D-K,  sexual  multi- 
plication; D,  microgametes;  E,  macrogamete;  F,  G,  fertilization;  #,  7,  K,  division 
,   and  spore  production.     (After  Schatidinn,  from  Doflein.) 

called  schizogony  and  the  young  parasites  are  called  merozoites,  if  a 
sexual  fertilization  has  not  immediately  preceded  the  act  of  division; 
if  such  a  division,  in  which  the  parent  organism  disappears,  takes  place 
after  a  fertilizing  act,  the  process  is  called  sporogony  and  the  young 
parasites  are  sporozoites. 


130  MORPHOLOGY   AND    CULTURE    OF   MICROORGANISMS 

In  protozoa,  as  in  metazoa,  the  essential  process  in  fertilization  is  the 
union  of  two  nuclei  of  opposite  sex.  In  dividing,  cells  may  go  through 
a  process  called  mitosis  during  which  the  chromatin  of  the  nucleus  is 
grouped  into  more  or  less  rod-shaped  masses  which  are  called  chromo- 
somes. The  number  of  chromosomes  which  are  formed  during  mitosis 
is  constant  and  characteristic  for  each  species.  In  the  reproductive 
areas,  during  the  two  divisions  just  preceding  the  maturity  of  cells 
which  are  to  become  ova  or  spermatozoa,  the  number  of  chromosomes  is 
reduced  to  exactly  one-half  of  the  number  which  are  formed  during  the 
division  of  cells  outside  of  the  reproductive  areas  of  the  same  animals. 
The  process  by  which  the  number  of  chromosomes  is  reduced  to  one-half 
is  termed  chromatic  reduction,  and  the  fragments  of  chromatin  which  in 
the  female  are  unused  and  which  are  extruded  from  the  cell  during  the 
process  are  called  polar  bodies.  While  reduction  in  the  number  of 
chromosomes  has  been  shown  to  occur  prior  to  fertilization  in  a  number 
of  the  protozoa,  in  many  species  a  more  primitive  process  consisting  of 
the  mere  extrusion  of  masses  of  chromatin  irrespective  of  the  number  of 
chromosomes  is  found  to  occur.  It  is  evident  that  the  chromatin  is, 
at  least  usually,  reduced  in  amount  preparatory  to  the  sexual  process. 

Although  in  certain  of  the  protozoa  nuclear  division  is  accomplished 
by  a  process  of  mitosis  similar  to  that  which  occurs  in  multicellular 
animals,  in  many  it  is  affected  by  a  much  more  primitive  process. 
The  nucleus  may  be  resolved  into  scattered  granules  of  chromatin — 
chromidia — which  may  subsequently  become  reconstructed  into  a  num- 
ber of  nuclei.  The  nucleus  may  divide  by  direct  division,  that  is,  by  sim- 
ple constriction  into  two  approximately  equal  parts.  Between  this  form 
of  division  and  the  classical  mitosis  there  is  every  possible  transition. 
The  centrioles  or  centrosomes  are  frequently  intranuclear  in  the 
protozoa.  In  the  case  of  primitive  nuclei  without  definite  nuclear  mem- 
brane a  division  simulating  mitosis  is  termed  promitosis.  In  other 
forms  in  which  there  is  a  nuclear  membrane  but  in  which  the  centrioles 
remain  intranuclear  throughout  division,  the  process  is  called  meso- 
mitosis.  The  nuclear  membrane  often  persists  throughout  division 
and  the  chromosomes  are  in  many  forms  very  minute  or  are  not 
definitely  formed. 

The  fertilizing  processes  which  occur  in  the  protozoa  may  be  grouped 
under  three  heads:  Copulation,  Conjugation  and  Self-fertilization.  In 
copulation  two  whole  cells  unite.  The  cells  taking  part  in  this  union 


PROTOZOA  131 

are  called  gametes  and  there  are  the  male  or  microgametes,  and  the 
female  or  macro  gametes.  The  cells  which  produce  the  gametes  are 
called  gametocytes.  The  product  of  the  union  is  called  a  copula  or 
zygote.  If  the  uniting  cells  be  equal  in  size  the  copulation  is  isogamous; 
if  they  be  unequal,  the  copulation  is  said  to  be  anisogamous.  Aniso- 
gamous  copulation,  the  union  of  two  unequal  cells,  is  most  typically 
seen  in  the  fertilization  of  a  large  macrogamete  by  a  small  microgamete. 
Copulation  is  the  most  common  fertilizing  process  among  the  patho- 
genic protozoa.  Conjugation,  the  second  method  of  fertilization,  only 
occurs  among  the  ciliata.  In  it,  two  adult  individuals  place  themselves 
in  apposition.  The  nucleus  of  each  cell  first  reduces  and  then  divides 
into  two  halves,  one  male,  the  other  female.  Each  organism  retains 
its  female  half  nucleus,  while  an  exchange  of  the  male  half  nuclei  is 
effected.  Processes  of  self-fertilization^  such  as  autogamy  and  partheno- 
genesis, are  included  under  the  third  heading.  In  autogamy  the  nucleus 
of  a  single  cell  divides  into  two  parts.  Each  of  these  may  undergo 
further  division,  during  which  the  chromosomes  are  reduced  or  there 
may  be  a  simple  extrusion  of  a  portion  of  the  chromatin.  The  two 
resulting,  reduced  nuclei  then  unite,  in  the  same  cell,  to  form  a  new 
nucleus.  Parthenogenesis  is  the  development  of  new  individuals  from  a 
female  cell  without  a  preceding  fertilization;  this  process  possibly  occurs 
in  many  protozoa,  and  through  it  perhaps  may  be  explained  the  reap- 
pearance of  malaria  in  patients  who  once  suffered  from  that  disease 
and  were  thought  to  have  recovered. 

The  LIFE  CYCLE  of  a  protozoon  consists  of  the  changes  through 
which  it  passes  in  the  period  intervening  between  each  fertilizing  act. 
In  many  of  the  pathogenic  protozoa,  an  alternation  of  generations 
occurs;  that  is,  cycles  of  development  in  which  an  asexual  method  of  re- 
production occurs,  alternate  with  cycles  of  development  in  which  re- 
production is  effected  by  sexual  methods.  The  developmental  cycles 
are  commonly  punctuated  by  binary  or  multiple  division,  by  encyst- 
ment,  and  by  transference  to  a  second  host  as  a  necessary  factor  for  the 
completion  of  the  life  cycle.  An  alternation  of  generations  occurs 
in  the  life  cycle  of  one  of  the  most  important  of  the  pathogenic  protozoa, 
the  parasite  which  produces  malaria  (Fig.  189).  While  it  is  in  the  body 
of  its  mammalian  host,  man,  it  multiplies  through  multiple  fission  or 
schizogony;  the  sexual,  or  propagative  phase  of  its  development 
occurs  within  the  body  of  its  invertebrate  host,  a  mosquito.  The 


132  MORPHOLOGY   AND   CULTURE    OF   MICROORGANISMS 

host  in  which  the  adult,  sexual  stages  of  the  parasite  occur,  in  this 
instance  the  mosquito,  is  said  to  be  the  definitive  host;  hosts  harboring 
the  parasite  while  it  is  in  other  stages  are  called  intermediate  hosts. 

ENCYSTMENT.— Under  unfavorable  conditions,  such  as  dry  surround- 
ings, many  protozoa  are  able  to  surround  themselves  by  a  resistant 
cyst  and  to  enter  upon  a  resting  stage  of  indefinite  length.  The  cyst 
protects  them  from-  harmful  influences  and,  surrounded  by  it,  they 
remain  in  a  resting  state  until  favorable  circumstances  come  about  once 
more.  The  power  of  forming  resistant  cysts  plays  an  important  part 
in  the  life  history  of  many  parasitic  protozoa;  it  is  especially  so  with 
those  protozoa  which  have  become  so  specialized  that  multiplication 
or  continuous  existence  independent  of  their  appropriate  host  has 
become  impossible  for  them.  It  is  often  through  the  formation  of 
cysts  that  an  infection  by  a  protozoon  is  spread,  and,  as  in  the  coccidia 
(page  889),  the  presence  of  such  a  stage  is  often  absolutely  essential 
in  the  life  history  of  a  parasite. 

PARASITISM 

A  parasite  is  an  organism  which  is,  at  some  time,  directly  dependent 
upon  another,  usually,  a  larger  organism. 

Although  the  word  parasite  is  often  used  as  though  it  referred  only 
to  organisms  belonging  to  the  animal  kingdom,  parasites  may  be 
either  animal  or  vegetable;  bacteria  and  fungi,  which  live  at  the 
expense  of  other  living  beings,  are  parasites  just  as  the  disease-pro- 
ducing protozoa  and  the  biting  insects  which  transmit  them  are 
parasites. 

Most  parasites  are  simple  organisms,  low  in  the  scale  of  life.  They 
nourish  themselves  without  exertion,  at  the  expense  of  their  hosts,  and 
as  might  be  expected,  their  unemployed  organs,  such  as  the  sensory 
locomotory  and  seizing  appendages,  by  means  of  which  food  is  usually 
obtained,  gradually  disappear;  degeneration  always  occurs  in  an 
organism  which  assumes  a  parasitic  mode  of  life. 

Organisms,  such  as  the  malarial  parasite,  which  are  wholly  de- 
pendent for  existence  upon  their  hosts,  are  called  obligatory  parasites; 
those  which  are  not,  such  as  the  infusoria  usually  found  in  the  stomach 
of  herbivorous  animals,  are  facultative  parasites.  Facultative  parasites 
often  feed  upon  organic  material  provided  by  the  host,  and  not  upon 


PROTOZOA  133 

the  host  itself;  but  they  are  capable  of  living  indefinitely  apart  from 
the  host. 

If  an  organism  is  attached  to  a  host,  and  neither  harms  nor  benefits 
it,  such  an  organism  and  its  host  are  said  to  be  commensals.  For 
example,  the  spirochsetes  found  about  the  teeth  of  many  persons  are 
usually  harmless ;  they  are  commensals  of  their  host.  When  the  host  of  an 
obligatory  parasite  dies,  the  parasite  often  perishes  also.  Consequently, 
it  is  contrary  to  the  interest  of  such  a  parasite  to  destroy  its  host;  yet 
parasites  often  do  harm  their  hosts.  The  harm  done  by  a  parasite  to  its 
host  is  the  disease  which  that  parasite  causes.  Disease  is  recognized  by 
symptoms.  The  nature  of  the  symptoms  depends  directly  upon  the 
nature  of  the  harm  done  by  the  parasite.  The  symptoms  are  the  result 
of  interference  by  the  parasite  with  tissues,  or  the  functions  of  tissues, 
in  the  host.  The  pathogenic  protozoa  may  injure  their  hosts  in  at  least 
three  ways :  They  may  feed  upon,  and  destroy  cells ;  they  may  produce 
poisonous  toxins;  and  their  presence  may  do  damage  by  mechanically 
obstructing  some  of  the  functions  of  its  host.  All  three  of  these  ways 
are  well  exemplified  by  the  action  of  the  malarial  parasite  in  man 
(page  892). 

DISCUSSION   OF   THE   C  L  AS  SIP  J  CATION* 

The  following  grouping  of  the  Protozoa  gives  a  general  idea  of  the 
position,  in  zoological  sequence,  of  the  individual  parasites  which  are 
spoken  of  in  the  subsequent  pages.  The  Protozoa  are  here  grouped 
in  four  classes:  the  RHIZOPODA,  the  FLAGELLATA,  the  SPOROZOA,  and 
the  INFUSORIA;  and  these  classes  are  divided  directly  into  genera.  This 
is  by  no  means  a  complete  classification  of  the  protozoan  families. 
Many  orders,  families  and  genera  are  unmentioned  because  they  are 
parasitic  neither  in  man  nor  in  animals;  and  of  the  organisms  mentioned, 
only  those  which  are  constantly  causes  of  disease  are  described. 

The  form  of  a  protozoon  may  vary  greatly  at  different  stages  of  its 
development;  for  example,  the  adult  herpetomonas  is  an  active  organism 
moving  by  means  of  a  flagellum,  quite  unlike  its  spherical  form  which 
is  without  a  flagellum.  Consequently,  the  whole  life  history  of  a  proto- 
zoon must  be  known  before  it  can  be  classified  with  absolute  certainty. 
The  whole  of  the  life  history  is  known  for  only  a  few  protozoa;.  and, 

•(See  p.   13.) 


134 


MORPHOLOGY  AND   CULTURE   OF   MICROORGANISMS 


though  the  organisms  mentioned  in  this  classification  are  placed  in 
the  position  usually  given  to  them,  it  must  be  understood  that  this 
classification  is  not  final,  and  that  the  discovery  of  new  stages  in  the 
life  history  of  some  of  these  protozoa  may  make  it  necessary  to  remove 
them  from  the  classes  in  which  they  have  been  placed.  For  example, 

before  its  flagellate  stage  was  known, 
Leishmania  donovani  was  classified  with 
the  sporozoa;  now  it  is  grouped  with  the 
herpetomonads. 

The  characteristics  of  the   different 
genera  and  of  the  unimportant  parasites 
are  very  briefly  mentioned  in  the  follow- 
11  ing  paragraphs;  the  important  parasites 

ifc|      I  are  treated  more  fully  in  the  pages  indi- 

lipli  cated  by  the  references  given,  in  brackets. 

ifeil      |l  The  RHIZOPODA  include  the  simplest 

forms  of  animal  life.  A  rhizopod,  such 
as  an  amoeba,  consists  of  a  single  cell, 
without  a  protective  covering,  and  with- 
out permanent  organs  of  locomotion;  it 
moves  about  and  captures  its  food 
through  the  agency  of  its  pseudopodia. 
Very  few  of  the  rhizopods  are  parasitic; 
most  of  those  which  are  parasitic,  belong 
to  the  genus  Entamceba.  Different 
species  of  parasitic  amoebae  may  occur 
in  the  alimentary  canals  of  various  ani- 
mals. Certain  of  these  produce  serious 
diseases  (page  876). 

The  FLAGELLATA  are  distinguished 
by  possessing  one  or  more  flagella; 
they  often  have,  also,  a  fin-like,  un- 
dulating membrane  extending  along  the  surface  of  their  body. 
Many  possess  two  nuclei,  a  larger  trophonucleus  which  has  to  do 
with  nutrition  and  a  smaller  kinetonucleus  which  is  intimately 
connected  with'  the  organs  of  locomotion.  This  group  has  been 
termed  the  Binucleata  by  certain  systematists.  Most  flagellates  are 
free-living.  Comparatively  few  species  are  parasitic,  but  some  of 
these  cause  very  serious  diseases  (page  879). 


B 


FIG.  95. — Herpetomonas 
musc(B-domestic<z  (Burnett).  A, 
Motile  individual  with  two  flag- 
ella; B,  cyst;  w,  nucleus;  bl, 
kinetonucleus.  (After  Pro- 
wazekfrom  Minchin.) 


PROTOZOA 


135 


A  Herpetomonas  is  an  elongated  organism  which  possesses  trophonu- 
cleus  and  kinetonucleus.  The  latter  is  situated  near  the  flagellar  or 
anterior  end  of  the  parasite,  and  from  it  arises  a  terminal  flagellum. 
A  Herpetomonas  has  no  undulating  membrane.  A  Crithidia  is  an  organ- 
ism like  a  Herpetomonas,  but  possessing  an  undulating  membrane. 
A  Trypanosoma  is  an  elongated  parasite  which  has  a  trophonucleus, 
a  kinetonucleus  usually  situated  near  its  aflagellar  extremity  and  an 


FIG.  96. — A,  Trypanosoma  tinea  of  the  tench;* note  the  very  broad  and  undulat- 
ing membrane  in  this  species;  B.,  C.,  T.  percce  of  the  perch,  slender  and  stout  forms. 
(After  Minchin,  X  2000.) 

undulating  membrane  along  the  border  of  which  the  flagellum  extends 
to  terminate  in  a  whip-like  appendage.  Species  of  Herpetomonas, 
Crithidia  and  Trypanosoma  are  frequently  found  in  the  intestines  of 
insects.  One  species  of  Herpetomonas  is  a  frequent  and  harmless  para- 
site in  the  intestine  of  the  house  fly.  Many  serious  diseases  are  caused 
by  trypanosomes.  The  genus  Try pano plasma  includes  organisms 
which  have  a  flagellum  at  either  end,  as  well  as  an  undulating  mem- 
brane. They  are  parasitic  in  the  blood  of  fishes.  The  genera  Cerco- 
monas,  Manas,  and  Plagiomonas  include  small,  unimportant  flagellate 


i36 


MORPHOLOGY   AND    CULTURE    OF   MICROORGANISMS 


organisms  which  have  been  found,  occasionally  in  the  human  intestine 
and  vagina,  and  in  necrotic  material  from  the  lungs.  Trichomonas 
is  a  pear-shaped  organism  which  has  four  flagella  attached  to  its  blunt 
end,  and  an  undulating  membrane  extending  from  the  origin  of  the 
flagella  at  the  anterior  end  posteriorly  over  the  surface  of  its  body. 


FIG.  97. — Trichomonas  eberthi,  from  the  intestine  of  the  common  fowl;  ///., 
anterior  flagella,  three  in  number;  P.fl.,  posterior  flagellum,  forming  the  edge  of  the 
undulating  membrane;  chr.  L,  "chromatinic  line,"  forming  the  base  of  the  undulating 
membrane;  chr.b.,  "chromatinic  blocks;"  bl.,  blepharoplast  from  which  all  four 
flagella  arise;  m.,  mouth  opening;  N.t  nucleus;  ax.,  axostyle.  (From  Minchin,  after 
Martin  and  Robertson.) 

One  of  the  four  flagella  is  usually  directed  backwards  and  extends  along 
the  border  of  the  undulating  membrane.  One  species  is  sometimes 
found  in  the  human  bladder.  Other  species  are  common,  usually 
harmless,  parasites  in  the  intestines  of  pigs,  frogs  and  other  animals. 
The  most  important  species  of  the  genus  Lamblia  is  Lamblia  intestinalis. 
It  also  is  a  pear-shaped  organism.  It  has  several  flagella  and  is  dis- 
tinguished by  possessing  a  depressed  sucker,  by  which  it  attaches  itself 


PROTOZOA 


137 


to  the  intestinal  epithelium  of  the  animal  in  which  it  lives.  It  is  a  cause 
of  diarrhoea  in  man,  and  also  of  a  fatal  disease  of  the  intestines  in 
rabbits;  but  it  is  almost  invariably  found  in  the  duodenum  and  first 
portion  of  the  small  intestine  of  normal  laboratory  animals  such  as 
mice,  rats,  and  rabbits. 


FIG.  98. — Lamblia  intestinalis.  A,  Ventral  view;  N.t  one  of  the  two  nuclei;  ax.> 
axostyles;^.1,^.2,  fl-3,fl-4,  the  four  pairs  of  flagella;  s.,  sucker-like  depressed  area  on 
the  ventral  surface;  x.,  bodies  of  unknown  function.  (After  Wenyon  (277)  from 
Minchin.) 

The  SPOROZOA  are  parasitic  protozoa  which  multiply  by  the  produc- 
tion of  spores  at  some  stage  of  their  life  cycle.  There  are  very  many 
sporozoa  and  so,  for  convenience  of  classification,  they  are  subdivided 
into  seven  orders.  The  Gregarina  have  ajvery  distinctive  shape;  the 
single  cell,  of  which  they  are  composed,  is  divided  into  two  or  more 
divisions.  The  first  of  these  divisions  is  furnished  with  hooks  or  other 
structures  through  which  the  parasite  attaches  itself  to  its  host.  None  of 
the  gregarines  are  parasitic  on  mammals;  worms  are  the  hosts  for  some 
of  them.  The  Coccidia  are  usually  parasitic  within  certain  cells  of  their 


138 


MORPHOLOGY  AND   CULTURE   OF   MICROORGANISMS 


host,  for  example,  Coccidium  stiedce  (Eimeria  cuniculi]  (page  889)  enters 
the  epithelium  of  the  small  intestine  and  of  the  bile  ducts  of  the 


*<& 


... 


FIG.  99. — Sporozoits  in  the  oocyst  of  Laverania  malaria.  A,  Formation  of 
nuclear  points  which  serve  as  the  foci  from  which  the  sporozoits  develop;  B,  a  more 
definite  shaping  of  protoplasm  and  nuclei;  C,  D,  mature  sporozoits  in  the  oocyst 
arranged  about  centers  from  which  they  radiate;  E,  a  portion  of  one  enlarged. 
(After  Grassiy  from  Doflein.} 

rabbit,  while  Eimeria  avium  enters  and  destroys  the  cells  lining  the 
intestines  of  the  birds  which  it  infects  (page  889).  The  Hamosporidia 
live,  for  a  part  of  their  life  cycle,  within  the  red  cells  of  the  blood  of 


PROTOZOA  139 

vertebrate  animals.  They  are  a  very  important  order.  The  genus 
Plasmodium  causes  malaria  in  man  (page  890) ;  while  Proteosoma  and 
Hamoproteus  are  malarial  parasites  of  birds  (page  890).  The  Hcemogre- 
garince  are  usually  harmless  parasites  of  reptiles  and  batrachians 
(frogs) ;  a  part  of  their  life  is  passed  within  the  red  cells  of  their  host, 
but  they  have  a  slowly  moving  stage,  somewhat  resembling  a  gregar- 
ine,  which  occurs  free  in  the  blood.  Hepatozoon  perniciosum  is  the 
best  known  of  a  group  of  haemogregarine-like  parasites  which  are 
parasitic,  often  within  the  white  cells  of  the  blood,  in  dogs,  in  rats,  and 
in  other  rodents;  so  far  as  is  known,  they  do  not  cause  disease.  The 
genus  Babesia  (page  894)  includes  parasites  which  cause  important 
diseases  in  cattle,  sheep,  horses  and  dogs.  Similar  parasites  have 
been  found  in  the  blood  of  monkeys,  of  dogs,  of  rats  and  other  rodents. 
The  Sarcosporidia  are  tube-like  in  shape  and  filled  with  spores.  They 
are  found  within  the  cells  of  the  voluntary  muscles.  TheHaplosporidia 
are  a  group  of  very  small  sporozoa  of  which  little  is  known.  Some  of 
them  are  parasitic  in  fish;  one  of  them,  Rhinosporidium  kinealyi,  has 
been  found  in  a  tumor  of  the  nose  of  a  native  of  India.  The  Myxo- 
sporidia  (page  899)  are  recognized  by  the  peculiar  form  of  their  spores; 
each  spore  has  one  or  more  capsules  each  furnished  with  a  coiled  fila- 
ment or  thread  which  is  extruded  under  certain  conditions  and  probably 
serves  to  anchor  the  spore  to  a  surface  upon  which  further  development 
may  occur.  Members  of  this  order  are  parasitic  in  various  tissues  of 
fishes  and  they  often  produce  disease  in  their  hosts.  The  spores  of  the 
Microsporidia  (page  899)  are  exceedingly  small;  a  member  of  this 
order  is  the  cause  of  pebrine  in  silk-worms  (page  937). 

The  INFUSORIA  (page  899)  are  a  large  class.  Most  of  them  are  not 
parasitic.  They  are  the  most  highly  developed  of  the  protozoa  and 
their  bodies  are  more  or  less  covered  with  cilia,  by  which  they  move 
themselves  through  the  liquids  in  which  they  live. 

Lastly,  under  the  heading  Parasites  of  Uncertain  Position,  are 
grouped  a  number  of  organisms  which  cannot  be  classified  because 
so  little  is  known  of  them  at  present.  The  spirochaetiform  organisms, 
Histoplasma  capsulatum  (page  900),  the  Chlamydozoa  (page  900),  the 
Rickettsias,  and  the  Ultramicroscopic  viruses  (page  119)  are  all  asso- 
ciated with  important  diseases  in  men  and  in  animals. 

The  SPIROCH^T^E  (page  900),  as  their  name  signifies,  are  thread-like 
organisms,  which  seem  to  be  coiled  in  a  spiral.  It  is  probable  that  the 


140  MORPHOLOGY   AND    CULTURE    OF    MICROORGANISMS 

curves  of  certain  spirochaetes  lie  in  one  plane  and,  consequently,  that 
their  bodies  are  really  waved  and  not  spiral.  These  organisms  have 
no  organized  nucleus.  The  chromatin  is  distributed  throughout  their 
bodies. 

Those  parasites  which  are  important  enough  to  require  special  con- 
sideration are  described  (page  876)  in  the  order  in  which  they  are  men- 
tioned in  the  classification  (page  13).  Whenever  it  is  possible  to  do  so, 
a  single  species  is  taken  as  the  type  of  each  genus  and  that  species,  with 
the  disease  it  produces,  is  described;  if  the  remaining  species  of  the 
genus  are  mentioned,  they  are  spoken  of  only  to  indicate  how  they 
differ  from  the  description  of  the  type. 

TECHNIC* 

The  methods  employed  in  studying  the  pathogenic  protozoa  are  very  similar  to 
those  used  in  bacteriology.  Microscopes,  with  the  highest  magnifications,  are 
essential  for  successful  work. 

It  is  of  great  importance  in  the  study  of  protozoa  to  examine  them  in  the  living 
condition.  In  no  other  way  can  their  mode  of  locomotion  be  determined  and 
frequently  their  contour  is  quite  different  in  living  and  in  fixed  preparations. 
A  small  amount  of  the  material  in  which  they  occur  may  be  placed  beneath  a  cover- 
glass  on  a  clean  slide  and  examined  immediately  with  the  microscope  by  ordinary 
daylight.  In  case  large  organisms  are  examined  in  rather  thin  fluid  it  is  well  to 
prevent  their  being  crushed  by  interposing  several  minute  globules  of  paraffin 
between  slide  and  cover-glass.  This  is  readily  accomplished  by  touching  paraffin 
with  a  hot  needle  and  transferring  it  thus  melted  to  several  points  on  the  slide  before 
the  preparation  is  made.  When  very  minute  forms  are  to  be  studied  it  is  necessary 
to  utilize  what  is  known  as  the  dark  field  illumination.  This  brings  out  very  minute 
organisms  and  particles  which,  being  transparent,  are  invisible  to  ordinary  trans- 
mitted light.  The  dark  field  apparatus  consists  of  a  strong  source  of  light  such  as  a 
small  arc  lamp,  a  special  condenser  which  deflects  the  light  so  that  objects  in  the 
microscopic  field  are  illuminated  by  light  directed  from  the  sides,  causing  them  to 
appear  bright  on  a  dark  background.  Another  method  of  obtaining  a  dark  field  is 
to  mix  on  a  slide  a  small  drop  of  the  material  to  be  examined  with  an  equal-sized 
drop  of  India  ink,  or  better  of  saturated  aqueous  solution  of  nigrosin,  and  then  to 
smear  this  mixture  across  the  surface  of  the  slide.  It  is  then  dried  and  examined  at 

*For  more  detailed  instructions  for  the  study  of  protozoa  see  Fantham,  Stephens  and 
Theobald,  The  Animal  Parasites  of  Man,  William  Wood  &  Company,  New  York;  Castellani 
and  Chalmers,  Manual  of  Tropical  Medicine,  Bailliere,  Tindall  &  Cox,  London;  Stitt,  Practical 
Bacteriology,  Blood  Work,  Parasitology,  Blakiston,  Philadelphia;  Brumpt,  Precis  de  Parasit- 
ologie,  Masson,  Paris;  Langeron,  Precis  de  Microscopic,  Masson,  Paris;  Doflein,  Lehrbuch  der 
Protozoenkunde,  Gustav  Fischer,  Jena;  and  Prowazek,  Der  mikroskopischen  Technik  der 
Protistenuntersuchung,  Leipzig. 


PROTOZOA  141 

once  by  the  oil  immersion  lens.  Only  ordinary  daylight  is  required  for  this  method 
but  it  does  not  serve  in  the  study  of  the  motility  of  organisms. 

By  special  apparatus  it  is  possible  after  obtaining  a  certain  amount  of  skill  to 
dissect  many  forms  of  protozoa.  In  this  way  knowledge  is  obtained  of  the  physical 
properties  of  various  portions  of  their  bodies  and  it  is  also  possible  to  inject  various 
chemicals  into  their  substance.  This  method  of  study  is  made  possible  by  the  me- 
chanical devices  utilized  by  Barber  to  whose  work  the  reader  is  referred.* 

In  order  to  make  stained  preparations  the  material  may  be  either  smeared  in  a 
thin  film  upon  clean  slides  or  sectioned  after  appropriate  treatment.  In  each  case 
the  material  requires  fixation.  For  the  preparation  of  stained  smears  the  Giemsa 
method  is  widely  used.  This  is  briefly  as  follows: 

1.  Make  thin  smears  of  material  on  a  clean  and  dry  slide. 

2.  Fix  immediately  by  covering  the  smear  with  pure  methyl  alcohol  which  should 
be  allowed  to  act  for  ten  to  twenty  minutes. 

3.  Dry  by  waving  slide  to  and  fro. 

4.  Stain  for  four  to  twenty-four  hours,  according  to  the  depth  of  stain  desired, 
in  a  solution  made  by  an  addition  of  one  drop  of  Giemsa  stain  to  i  c.c.  of  distilled 
water. 

5.  Rinse  with  distilled  water. 

6.  Dry  and  mount  in  immersion  oil  or  any  acid-free  balsam. 

It  is  frequently  desirable  to  keep  stained  smears  unmounted  as  they  apparently 
retain  their  color  for  a  longer  period  of  time.  They  may  be  studied  with  the  oil 
immersion  lens  but  the  oil  should  at  once  be  rinsed  off  with  xylol,  for  if  left  upon  the 
preparation  an  insoluble  substance  is  formed  which  produces  a  clouded  appearance. 
All  stained  preparations  should  be  stored  away  from  the  light  when  not  in  use.  For 
the  above  method  it  is  important  to  have  all  glassware  perfectly  clean  and  without 
trace  of  acid.  The  stain  must_  be  used  immediately  after  preparation.  Certain 
materials  may  be  smeared  very  readily  with  the  platinum  loop  ordinarily  used  in 
bacteriology.  A  very  practical  method  for  making  blood  smears  is  to  gather  a 
minute  drop  of  freshly  drawn  blood  from  a  small  needle-prick  of  the  skin  on  one 
edge  of  the  end  of  a  slide.  The  latter  is  placed  in  contact  with  the  surface  of  another 
slide  and  being  held  at  an  angle  of  45  degrees  is  pushed  steadily  lengthwise  across  its 
surface.  By  increasing  or  decreasing  this  angle  a  thicker  or  thinner  film  may  be 
made.  Certain  investigators  prefer  to  use  what  is  termed  the  wet  method  for  the 
fixation  of  smears.  In  this  case  the  smear  is  dropped  face  down  immediately  and 
before  drying  into  a  fixative  composed  of  two  parts  of  a  solution  of  saturated  HgCU 
in  distilled  water  and  one  part  of  absolute  alcohol.  The  technic  -employed  in  the 
staining  of  sections  is  then  followed  and  the  smear  is  not  allowed  to  dry  at  any  step 
in  the  procedure. 

The  preparation  of  stained  sections  requires  a  considerable  amount  of  technical 
skill.  Tissue  is  first  fixed  to  render  its  structure  permanent.  It  is  then  dehydrated 
in  alcohol  of  increasing  strengths,  next  placed  in  chloroform  or  some  other  clearing 
reagent  and  it  is  then  imbedded  in  paraffin,  after  which  it  may  be  sectioned.  For 

*Barber:  University  of  Kansas,  Science  Bulletin  1907-4-3;  also  Journal  of  Infectious 
Diseases,  1911,  8,  248,  an4  1911,  9,  117, 


142  MORPHOLOGY   AND   CULTURE    OF   MICROORGANISMS 

the  details  of  sectioning  and  the  staining  of  sections  the  reader  is  referred  to  Mallory 
and  Wright's  Pathological  Technic,  W.  B.  Saunders  and  Co.,  and  to  Lee's  Vade 
mecum. 

The  cultivation  of  free-living  protozoa  is  usually  accomplished  by  keeping  a 
supply  of  the  medium  in  which  they  live  on  hand.  Hay  infusion  prepared  by  boiling 
a  quantity  of  chopped  hay  in  water  is  an  easy  and  valuable  method  of  preparing 
culture  media.  For  the  cultivation  of  amoebae,  the  following  media  is  widely  em- 
ployed. It  should  be-noted,  however,  that  the  amoebae  which  have  been  cultivated 
are  regarded  as  free-living  forms  and  the  attempts  to  cultivate  parasitic  amoebae 
have  thus  far  been  unsuccessful. 

MEDIUM  OF  MUSGRAVE  AND  CLEGG 

Agar 20  to  30  g. 

Liebig's  extract  of  beef 3  to  .  5  g. 

Common  salt 3  to  .  5  g. 

Water 1,000  c.c. 

This  medium  is  designed  to  provide  for  slow  bacterial  growth  in  order  to  provide 
food  for  amoebae.  On  a  richer  medium  the  latter  are  overwhelmed  by  the  rapid 
growth  of  bacteria. 

For  the  cultivation  of  trypanosomes,  leishmania  and  other  flagellates  the  so- 
called  triple  N  media  is  employed.  This  is  prepared  as  follows: 

NlCOLLE,    NOVY',    MACNEAL   MEDIUM 

Water 900  c.c. 

Salt 6  g. 

Agar 16  g. 

Dissolve,  distribute  in  tubes,  sterilize  and  add  to  the  medium  in  each  tube  after 
liquefying  and  cooling  to  4o°-5o°C.  one-third  its  volume  of  rabbit  blood  obtained  by 
cardiac  puncture.  Slope  the  tubes  for  twelve  hours,  incubate  at  37°  for  five  days 
to  prove  the  sterility  of  the  medium  and  then  keep  them  at  the  ordinary  temperature 
of  the  laboratory  for  a  few  days  before  sowing  them.  (The  tubes  should  be  sealed  to 
prevent  evaporation.) 

The  malaria  organisms  have  been  made  to  continue  development  outside  the  body 
by  the  following  method  devised  by  Bass. 

Bass's  Method. — The  blood  in  10-  to  2o-c.c.  quantities  is  taken  from  the  patient's 
vein  and  received  in  a  centrifuge  tube  which  contains  }{  Q  c,c.  of  50  per  cent,  glucose 
solution.  A  glass  rod,  or  piece  of  tubing,  extending  to  the  bottom  of  the  centrifuge 
tube  is  used  to  defibrinate  the  blood.  After  centrifugalizing  there  should  be  at  least 
i  inch  of  serum  above  the  cell  sediment.  The  parasites  develop  in  the  upper  cell 
layer  about  Ko  to  3^o  inch  from  the  top.  All  of  the  parasites  contained  in  deeper 
lying  red  cells  die.  To  observe  the  development,  red  cells  from  this  upper 
portion  are  drawn  up  with  a  capillary  bulb  pipette. 


PROTOZOA  143 

Should  the  cultivation  of  more  than  one  generation  be  desired,  the  leucocyte 
upper  layer  must  be  carefully  pipetted  off,  as  the  leucocytes  immediately  destroy  the 
merozoites.  Only  the  parasites  within  red  cells  escape  phagocytosis.  Sexual 
parasites  are  much  more  resistant,  and  the  authors  think  they  observed  partheno- 
genesis The  temperature  should  be  from  40°  to  41°  and  strict  anaerobic  conditions 
observed.  ^Estivo-autumnal  organisms  are  more  resistant  than  benign  tertian  ones. 
Dextrose  seems  to  be  an  essential  for  the  development  of  the  parasites. 

Noguchi  first  cultivated  spirochsetes;  others  have  extended  and  modified  his 
work.  If  a  few  drops  of  blood  containing  spirochaetes  is  dropped  into  sterile  ascitic 
fluid,  hydrocele  fluid,  or  blood  plasma,  to  which  a  piece  of  sterile  tissue,  such  as 
rabbit's  kidney,  is  added,  the  spirochaetes  multiply.  The  test  tube  should  contain 
about  15  ccm.  of  culture  fluid.  It  is  advantageous  to  cover  the  fluid  with  sterile 
paraffin  oil. 


PART  II 
PHYSIOLOGY  OF  MICROORGANISMS 


DIVISION  I 


INTRODUCTION* 

Microbial  physiology  seeks  to  understand  the  material  or  concrete 
processes  and  functions  of  protoplasmic  activity  which  integrate  in  the 
phenomenon  of  life.  They  are  embodied  in  some  form  of  an  organism. 
The  many  assembled  and  harmonious  forces  involved  in  a  unit  of  life, 
while  they  may  be  resolved  in  a  degree  elementally,  are  dependent  upon 
the  structure,  composition,  and  energy  values  of  the  life-form,  and  also 
upon  its  environment;  likewise  the  reverse  is  true.  The  concomitant 
relations  of  forces  to  the  life-form  and  life-form  to  the  forces  are  more  or 
less  hidden  at  present,  yet  they  are  slowly  becoming  apparent. 

Owing  to  the  multiplicity  and  variety  of  forces  operating  in  physio- 
logical functioning,  it  is  patent  that  physiology  is  complexly  composite 
in  nature  and  must  resort  to  the  elemental  branches  of  science  as 
physics,  chemistry  and  morphology  for  its  understanding  and  exposi- 
tion. Shrouded  along  with  demonstrated  knowledge  is  the  mysterious 
veil  of  life  which  makes  of  it  a  reality,  subject  to  the  ready  onslaught 
of  scientific  attack  and  to  a  spirit  which  halts  approach. 

Besides  the  basis  of  facts  found  in  physics,  chemistry  and  morphol- 
ogy which  contribute  freely  to  the  structure,  there  are  the  immediate 
matters  of  cytology,  anatomy  both  gross  and  histological,  and  environ- 
mental conditions  which  lead  into  fields  of  essential  technic,  before 
physiology  can  be  truly  grasped  or  successfully  studied.  When  the 

*  Prepared  by  Charles  E.  Marshall  and  Arao  Itano. 
10  145 


146  PHYSIOLOGY  OF  MICROORGANISMS 

study  is  attempted  in  an  elementary  manner  it  consists  in  recognizing 
observational  functions  without  attempting  to  explain  or  understand 
the  mechanism  frehind.  Such  attempts  belong  to  the  first  steps  in 
primary  and  secondary  education.  The  limitations  or  boundaries  of 
physiological  knowledge  are  those  established  by  the  human  mind  in 
laying  hold  of  and  utilizing  the  nature  and  the  facts  of  the  elemental 
studies  upon  which  physiology  rests  and  its  ability  to  translate  them  in 
the  life-mechanism  at  work. 


CHAPTER  I 
THE  UNIT  OF  BIOLOGICAL  ACTIVITY* 

Whether  a  cell  acts  in  the  capacity  of  an  individual  entity  or  alone, 
as  in  the  case  of  S.  ceremsi(B)  or  in  its  intimate  association  with 
other  cells  possessing  an  individual  entity  and  having  an  intercellular 
relationship  as  yeasts  and  acetic  bacteria,  or  even  in  close  dependent 
relationship  with  other  cells  in  forming  a  multicellular  entity  as  in  the 
case  of  metazoa  and  metaphytes,  its  self-functioning  processes  or  its 
performances  are  confined  within  the  limits  of  the  cell,  are  the  operating 
mechanism  of  the  cell  and  are  independent  of  other  cells,  notwithstand- 
ing the  controlling  influences  of  environmental  conditions  upon  its 
activities.  The  self-contained  cell,  which  is  a  cellular  entity,  may  not 
be  subject  as  a  rule  to  the  more  immediate  influence  of  other  cells,  yet 
the  associative  influence  exists  in  most  cases  whether  near  or  remote 
and  is  more  or  less  essential  to  the  life  of  the  cell.  There  is,  in  other 
words,  a  biological  interdependence  in  most  living  forms.  It  follows 
that  the  cell  has  therefore  a  distinct  life  of  its  own  within  its  sphere 
of  activity  and  in  addition  an  equally  important  office  to  perform  in  its 
association  with  other  cells;  in  both  cases,  it  functions  only  together 
with  its  environment. 

The  cell  is  at  the  mercy  of  environmental  conditions.  S.  ceremsia 
is  master  of  itself  until  the  factors  of  food,  moisture,  respiration, 
temperature  and  reaction  are  demanded  for  the  sustenance  of  life;  it 
then  becomes  the  menial  servant  of  each,  for  each  and  every  factor  is 
essential  to  its  existence.  In  turn,  food,  required  gases,  temperature, 
and  other  environmental  factors,  such  as  may  be  needed  for  cell  life, 
may  have  their  source  in  the  activities  of  other  cells.  Yeast  cells 
produce  alcohol  for  the  acetic  bacteria  as  certain  organs  of  the  body 
produce  hormones  for  the  activity  of  other  organs.  One  cell,  therefore, 
through  external  factors  may  become  dependent  upon  another  and 
probably  is  in  the  case  of  most  cells. 

Unless  the  microorganisms  which  seemingly  live  directly  upon  the 
very  simple  elements  of  nature  are  excluded,  cellular  life  is  so  intricately 

*  Prepared  by  Charles  E-  Marshall  and  Arao  Itano. 

147 


148  PHYSIOLOGY   OF   MICROORGANISMS 

bound  up  with  other  cellular  life  that  it  ceases  without  such  association, 
whether  it  is  regarded  from  the  standpoint  of  individual  entities,  the 
protophytes  and  the  protozoa,  or  the  standpoint  of  the  complex  entities, 
the  metaphytes  and  metazoa.  Virchow  made  the  cell  the  working  unit 
system  of  life,  but  it  was  done  in  the  sense  that  the  "  House  of  Roths- 
child" has  become  a  unit  in  the  financial  world.  Pfliiger,  Verworn, 
Ehrlich  and  Vaughan,  however,  resolve  the  cell  or  the  " House"  into 
the  ultimate  coordinated  agencies  within,  molecular  complexes,  which 
are  responsible  for  the  inception  and  continuing  of  all  the  activities. 
There  are  cells,  it  is  true,  of  many  kinds  and  different  degrees  of  struc- 
ture and  organization,  accordingly  a  more  elemental  unit  must  be 
sought  which  is  essential  to  establish  harmony  or  unity  in  life's  ultimate 
phenomena  or  reactions.  Vaughan,  who  is  the  last  of  the  above  to  write 
from  his  own  investigations,  says:  "The  cell  is  not  the  unit  of  life;  life 
is  molecular.  Life  is  function,  not  form."  Again  he  says:  "Cells 
consist  of  a  chemical  unity  made  of  giant  molecules."  Moore  states 
that  "the  unit  of  the  biologists  is  the  living  cell,"  but  he  himself 
approaches  it  from  the  standpoint  of  molecular  structure.  He  would 
impugn  the  attitude  and  circumscribe  the  field  of  the  biologist  by  the 
limits  of  morphology  whereas,  in  fact,  the  biologist  interprets  organic 
life  by  means  of  the  various  ultimate  elements  included  so  far  as  this  is 
possible  and  endeavors  to  unify  all  forces  and  structures  in  an  inti- 
mate unity. 

Physiology  in  taking  cognizance  of  the  cell  itself  and  its  environment 
is  reduced  to  its  simplest  and  lowest  terms  in  the  cell  possessing  an 
individual  entity,  for  a  large  part  of  this  physiology  is  found  represented 
in  its  most  rudimentary  and  elemental  forms  and  consequently  quite 
easily  studied. 

Nutrition  as  it  is  illustrated  in  B.  subtilis  is  easily  approached  as 
compared  with  that  of  man.  It  is  not  difficult  to  reproduce,  change, 
and  control  nutritional  conditions  in  a  unicellular  organism  as  compared 
with  the  multicellular  organisms.  Methods  which  enable  the  investi- 
gator to  reproduce,  manipulate  and  supervise  microorganisms  enable 
him  to  attack  problems  excluded  from  the  category  of  the  multicellular 
physiologist.  However,  the  physiologist  of  complex  forms,  as  the 
human,  not  only  has  his  problems  rendered  more  intricate  by  the  organ- 
isms he  studies  but  he  also  has  them  multiplied  because  of  the  many  fold 
combinations  of  cells.  Bayliss  is  substantially  correct  when  he  says, 


THE    UNIT    OF   BIOLOGICAL   ACTIVITY  149 

"The  physiology  of  unicellular  organisms,  although  of  considerable 
importance,  is  not  to  be  regarded  as  a  general  physiology,"  because  the 
physiology  of  microorganisms  leads  only  to  the  point  where  one  phase 
rule  must  give  way  to  another  phase  rule — the  physiology,  in  fact,  of 
any  restricted  number  of  organisms  harmoniously  related  can  never 
cover  the  field  of  general  physiology.  Let  us  examine  this  more 
particularly  for  the  purpose  of  securing  the  most  effective  attitude  in 
the  study  of  microbial  physiology. 

The  differences  which  separate  unicellular  physiology  from  the 
specific  human  physiology  are  worthy  of  consideration.  Neither  the 
one  nor  the  other  can  be  considered  general  or  comparative  physiology 
but  both  have  values  which  are  of  interchangeable  advantage.  The 
unicellular  organism  enters  upon  its  nutritional  career  as  a  free  cell 
found  in  an  environ  of  food  which,  in  its  specific  manner,  it  must 
prepare  for  absorption  and  assimilation.  The  human  first  brings  its 
food  into  a  canal,  a  tube,  the  alimentary  canal,  lined  with  specialized 
cells  which  contribute  to  the  preparation,  the  absorption  and  assimila- 
tion of  the  food.  Enzymes  are  secreted  by  the  unicellular  form  into  the 
food  medium  undergoing  preparation  for  absorption,  just  as  enzymes 
are  secreted  by  the  cells  of  the  walls  of  the  stomach  and  intestines  of 
the  human  species.  The  same  purpose  is  apparent  in  each  case.  The 
food  is  absorbed  through  the  cell-wall  or  directly  into  the  protoplasm  in 
the  unicellular  organism,  while  in  man  the  cells  lining  the  alimentary 
tract  operate  in  much  the  same  manner.  In  the  human  the  distribution 
takes  place  by  means  of  a  carrier  system,  the  circulation,  in  order  to 
reach  the  distant  points  while  with  the  single-celled  organisms  the  process 
is  one  of  diffusion.  Enzymes  are  present  in  both  organisms  engaged  in 
the  process  of  converting  food  material  into  protoplasm  and  the 
production  of  waste.  Aside  from  the  distributive  method,  the  nutritive 
processes  are  much  the  same  in  both.  It  now  remains  to  conclude 
which  is  the  more  simple  to  study,  the  more  accessible  for  study,  and 
the  more  adaptable  to  study.  In  this  the  single-celled  organism  has 
many  advantages.  This  does  not  constitute,  however,  general  or 
comparative  physiology  for  either  one  of  these  assumes  a  large  number 
and  a  varied  number  of  living  forms  into  which  creep  specific  differences. 

The  similarity  paralleled  in  nutrition  could  be  carried  into  other 
functions  but  this  is  not  the  purpose.  It  is  desirable,  mainly,  to  em- 


150  PHYSIOLOGY   OF   MICROORGANISMS 

phasize  the  possible  extension  of  the  simple  and  even  rudimentary 
basic  facts  to  the  field  of  general  physiology. 

The  complicated  structures  of  the  metazoa  and  metaphytes  can 
scarcely  be  compared  with  microorganisms.  It  is  difficult  to  speculate 
intelligently  on  the  development  of  shape  and  structures  in  biological 
forms  in  the  light  of  present  knowledge,  yet  proceeding  from  simple 
forms  to  the  more  complex,  there  is  constantly  confronting  the  mind 
the  possibility  on  the  part  of  nature  to  adjust  form  and  structure  to  the 
growing  and  expanding  demands  of  protoplasm.  The  struggle  on  the 
part  of  a  microorganism  to  secure  oxygen  or  to  get  away  from  it,  the 
action  of  light  and  darkness  on  the  growth  of  molds  and  other  factors 
signify  as  much  to  a  simple  single-celled  organism  as  the  prehensile 
tendencies  of  an  insect  or  an  ape.  It  is  something  sought  by  the  use  of 
different  structures  and  of  different  agents.  There  are  so  many 
indications  of  incipient  developments  in  the  unicellular  organism  that 
much  time  and  space  could  be  given  to  speculative  possibilities.  Only 
a  suggestion  is  required,  however,  to  start  the  thinking  student  to 
fruitful  reflection.  The  morphologist  approaches  the  subject  of  the 
place  of  microorganisms  in  nature  through  the  channels  of  "  degenerated 
forms"  from  "higher  forms"  or  "types  of  simple  forms  in  process  of 
evolution."  This  view  does  not  harmonize  with  the  physiological 
approach  in  which  functioning  supercedes  form  unless  degeneration 
and  evolution  is  made  a  matter  of  functioning  instead  of  form. 

A  cell  as  a  free  unit  and  a  cell  as  a  unit  but  tied  into  a  community 
of  cells  awaken  interest.  The  free  cell  might  be  likened  to  primitive 
man,  a  Jack-of -all- trades,  but  a  cell  interwoven  into  a  multicellular 
organism  is  that  of  a  Jack-of -all -trades,  an  individual,  and  a  man  in  a 
social  organization,  a  specialist.  This  seems  to  be  apparently  true 
except  where  the  cells  simply  form  an  aggregation  or  a  colony  in  which 
all  cells  function  alike.  There  is,  too,  the  tendency  of  the  single  free 
cell  to  specialize  as  those  of  the  alcoholic  group,  the  lactic  group  and 
many  others.  To  say  that  they  are  in  the  process  of  functioning  to- 
gether in  an  organism  as  the  yeast  and  acetic  organisms  is  wholly 
chimerical  yet  very  suggestive  in  nature's  slow  evolutions.  Few  facts 
can  be  brought  forward  to  disentangle  definitely  such  conceptions. 

As  a  working  basis  to-day  it  is  necessary,  because  of  restricted 
human  advancement,  to  consider  the  cell  as  the  unit  of  life  although  its 
compositional  structure  when  understood  may  reveal  the  unit  of  life 
for  to-morrow. 


THE   UNIT   OF  BIOLOGICAL  ACTIVITY  151 

THE  MECHANISM  OF  CELLS 

In  the  early  stages  of  this  book  Guilliermond  has  revealed  the  struc- 
tures of  microbial  cells  and  has  indicated  in  his  treatment  the  probable 
relations  of  these  structures  to  functioning.  The  immediately  fore- 
going section  carries  the  suggested  creative  powers  of  the  various  cell- 
structures  to  molecular  foci  which  seem  to  be  the  centers  in  which  vital 
changes  are  occurring.  It  now  remains  to  set  forth  the  mechanism  of 
the  cell,  functioning  as  a  whole,  which  can  be  done  only  by  approaching 
it  through  the  channels  of  its  activities. 

A  living  organism  is  conspicuous  as  a  mechanism  because  it  has  the 
power  of  self-maintenance  in  the  presence  of  a  suitable  environment, 
it  can  reproduce  its  like  when  conditions  are  favorable,  it  frequently  has 
the  freedom  of  motion,  and  possesses  many  reacting  responses  to  stimuli 
or  expressions  of  dynamic  existence.  These  are  characteristics  which 
belong  peculiarly  to  cell-life. 

With  food  at  hand  and  other  favorable  conditions  the  cell  becomes 
an  automaton.  The  material  needed  in  growth  and  the  energy  required 
for  operation  are  obtained  without  assistance  and  regulated  to  meet  the 
exact  demands  of  the  cell.  If  food  is  plentiful,  growth  and  reproduction 
reach  out  to  conserve  it;  if  food  is  scarce,  the  cell  accommodates  itself  to 
its  limitations  by  reduced  activity  or  a  resting  stage.  This  great  power 
of  adjustability  while  having  boundaries,  is  as  useful  to  the  life  of  the 
cell  as  it  is  wonderful.  Likewise,  if  solid  food  alone  is  available,  the 
food  is  reduced  to  a  liquid  form  or  changed  that  it  may  be  incorporated 
in  this  bit  of  protoplasm,  the  cell.  When  there,  it  is  transmuted  into 
the  living  substance  or  is  converted  from  dead  matter  to  living  matter. 
In  performing  this  work,  the  food  consumed  has  had  to  provide  the 
energy  as  well  as  the  material  which  rejuvenates  the  protoplasm,  for 
this  constant  rejuvenating  process  in  the  case  of  protoplasm  seems 
essential  to  life  as  well  as  to  the  constant  supply  of  energy  to  make  it 
possible. 

There  are  agents,  or  properties,  or  substances  of  the  cell  which  make 
this  possible,  it  is  true,  and  there  are  structures  of  the  cell  which  are 
involved  in  it,  but  these  are  necessarily  subordinate  to  the  innate  proto- 
plasmic forces  which  give  them  birth  and  which  in  some  manner  create 
and  supervise  them.  These  detailed  features  are  the  subjects  of  physi- 
ological study. 


152  PHYSIOLOGY   OF   MICROORGANISMS 

Unicellular  organisms  are  concerned  with  what  may  be  called 
respiration.  Oxygen,  in  its  free  forms,  is  needed  in  most  instances  for 
body-processes  and  carbon  dioxide  is  given  out.  The  amount  required 
varies  with  different  organisms  and  is  not  determined  by  the  amount 
present  in  the  air.  Other  gases  are  also  used.  Nitrogen  is  taken  up 
by  the  microorganisms  which  grow  on  the  roots  of  legumes;  marsh-gas, 
carbon  monoxide,  carbon  dioxide,  hydrogen  and  other  gases  are  claimed 
to  be  utilized.  Each  species  of  microorganisms  seems  to  possess  greater 
or  less  specificity  in  its  gas-relations.  No  species  can  extend  its  influ- 
ence over  all  amounts  of  a  single  gas  or  to  all  gases.  From  this  situation 
it  is  a  forced  conclusion  that  all  cells  do  not  function  alike,  accordingly 
must  have  different  mechanisms  although  the  capacity  for  life  appears 
common  to  all. 

Some  organisms  emit  light  or  manifest  radiant  energy,  other  organ- 
isms produce  pigments  which  vary  with  different  species,  still  others 
elaborate  deadly  poisons,  called  toxins,  and  other  substances  of  patholog- 
ical significance.  Briefly,  microorganisms  give  rise  to  many  different 
products  whether  they  take  the  form  of  secretions,  excretions,  energy 
manifestations,  or  are  referable  to  action  upon  environmental  media. 
Enumeration  is  not  the  purpose  here.  These  very  numerous  products 
indicate  the  many-sided  activities  of  microbial  life.  If  the  substances 
which  enter  a  mechanical  device  are  the  same,  the  products  passing  out 
are  the  same.  In  this  case,  the  food  substances  and  the  conditions 
controlling  the  microorganisms  may  be  identical  yet  the  issuing  products 
are  different.  It  follows,  therefore,  that  the  mechanism  of  cells  must 
be  very  different,  yet  as  mentioned  heretofore  the  life-mechanism  is 
common  and  the  same  so  far  as  determinable. 

Many  unicellular  forms  have  a  cell-wall  and  when  no  cell-wall  is 
present  there  is  likely  to  be  a  distinctive  and  denser  layer  of  cytoplasm 
on  the  outer  zone.  Some  organisms  when  placed  in  a  dense  salt 
solution  will  lose  a  considerable  amount  of  their  water-content  and 
shrink  while  others  will  not;  in  other  words,  some  microorganisms  have 
the  power  of  withstanding  dense  brine  while  others  do  not.  Osterhout 
has  repeatedly  demonstrated  that  where  two  salts  are  together  in  a 
medium  acting  as  a  substratum  for  microorganisms  there  may  exist 
an  antagonistic  or  favorable  action  of  one  of  the  salts  upon  the  absorp- 
tion of  the  other  salt  by  the  cell  but  that  cells  vary  in  these  responses. 

It  is  a  common  knowledge  that  cells  have  a  selective  action  which 


THE    UNIT    OF   BIOLOGICAL   ACTIVITY  153 

differs  with  different  microorganisms.  The  capacity  to  utilize  certain 
foods  in  the  presence  of  others,  to  acquire  certain  ingredients,  and  to 
leave  others  behind  makes  for  great  variability  in  the  functioning  of 
microbial  cells. 

Spores  of  some  microorganisms  are  exceedingly  resistant,  with- 
standing over  twenty  hours  of  heat  at  100°.  Spores  of  other  species 
are  destroyed  in  from  one  to  five  minutes  at  100°  under  the  same  con- 
ditions. Wide  differences  are  found  among  vegetative  cells  likewise. 
The  molecular  stability  which  embodies  the  life  of  the  cell  against  heat 
must  be  as  variable  as  the  dissolution  of  chemical  substances  under 
the  influence  of  heat.  Life  then  can  be  associated  with  many  kinds  of 
molecular  mechanisms  although  for  each  species  there  is  a  more  or 
less  constancy. 

In  the  process  of  selecting,  digesting  and  assimilating  their  food, 
certain  microorganisms  develop  an  unusual  amount  of  heat  so  much  that 
many  microorganisms  cannot  live  in  its  presence,  yet  these  heat- 
producers  (thermophile  microorganisms)  are  dependent  upon  this 
excessive  heat  for  their  proper  growth  and  functioning. 

So  far  evidences  of  varied  processes  existing  in  different  species 
and  strains  have  been  set  forth  in  the  products  and  energy  resulting 
from  growth  and  development,  or,  more  briefly,  in  the  processes  of 
metabolism.  These  clearly  convey  the  very  noticeable  variation  which 
must  exist  in  the  mechanisms  which  are  responsible  for  such  differences. 

There  is  another  approach  which  will  contribute  force  to  what  has 
already  been  said  concerning  the  mechanisms  of  living  cells. 

Chemists  have  repeatedly  shown  that  cells  differ  in  their  chemical 
compositions.  When  chemists  begin  their  destructive  laboratory 
manipulations  for  the  purpose  of  ascertaining  what  is  present  in  a  cell, 
they  may  not  be  working  with  the  real  substances  or  bodies  making  the 
living  protoplasm.  However,  the  bodies  which  they  do  study  and 
which  are  fairly  constant  in  the  same  kind  of  cells  or  in  the  same  species 
are  doubtless  products  which  in  one  form  or  another  enter  into  the 
complexity  of  protoplasm.  The  chemical  substances  studied  in  nervous 
tissue  differ  from  those  of  muscles;  the  chemical  substances  as  products 
of  certain  species  of  microorganisms  differ  from  the  products  of  other 
species.  Some  of  these  substances  are  definitely  known  to  differ  and 
others,  although  they  cannot  be  satisfactorily  determined,  are  suspected 
to  differ  in  different  tissues  and  different  species  of  microorganisms. 


154  PHYSIOLOGY   OF   MICROORGANISMS 


A  specific  and  more  definite  consideration  of  this  subject  will  appear 
later.  Our  purpose  here  is  to  point  out  simply  that  the  material  making 
up  the  molecular  mechanism  of  the  cell  must  be  exceedingly  delicate 
and  complex  because  of  its  products,  and  the  mechanism  of  one  cell 
must  differ  very  significantly  from  that  of  another  also  because  of  its 
products,  which  as  cell-contents  are  determinable;  and  further,  the 
mechanism  must  be  highly  responsive  because  of  its  ready  reactions  to 
various  influencing  agents.  All  of  these  evidences  seem  to  accord 
with  the  interpretation  advanced  in  the  previous  chapter — the  cell 
consists  of  chemical  foci  and  physical  forces  harmoniously  constructed 
into  an  operating  mechanism,  the  protoplasm,  which  is  arranged  effec- 
tively in  a  cellular  laboratory,  the  cell. 

Literature  freely  consulted  and  recommended  for  extended  study : 
BAYLISS,  The  Principles  of  General  Physiology. 
CHILD,  Individuality  in  Organisms. 
CZAPEK,  Chemical  Phenomena  in  Life. 
HENDERSON,  The  Fitness  of  the  Environment. 
MOORE,  The  Origin  and  Nature  of  Life. 

VAUGHAN,  Protein  Split  Products  in  Relation  to  Immunity  and  Disease. 
VERWORN,  General  Physiology. 


: 


CHAPTER  II 

A  STUDY  OF  PHYSICAL  FORCES  INVOLVED  IN  BIOLOGICAL 

ACTIVITIES* 

INTRODUCTION 

It  is  becoming  more  and  more  evident  that  besides  the  chemical 
components  and  forces  which  constitute  the  mechanism  and  activities 
of  the  cell  and  which  will  be  treated  in  the  next  chapter,  there  are 
certain  physical  forces  and  conditions  as  important  and  very  depen- 
dently  related  which  are  operative.  In  the  physical  or  chemical  ap- 
proach to  physiology  the  writers  do  not  assume  the  role  of  being 
chemists  or  physicists  and  they  write  somewhat  hesitatingly  and  reluc- 
tantly upon  such  matters,  yet  the  stream  separating  the  various 
branches  of  science  must  be  spanned.  The  purpose,  as  writers,  will  be, 
therefore,  to  bring  the  elementary  gist  of  the  physical  laws  and  phenom- 
ena, which  bear  upon  microbial  physiology,  to  the  attention  of  the 
student  for  memory-helpfulness  and  suggestiveness.  Should  greater 
knowledge  or  more  extensive  reading  be  desired  the  student  is  asked  to 
consult  the  literature  appended  at  the  end  of  this  chapter. 

ENERGY 

Energy  may  be  most  effectively  presented  in  the  general  law 
through  which  it  defines  itself  and  governs  applications,  and  in  the 
specific  expressions  in  applications  which  provide  the  detailed  back- 
ground. Energy  is  work  or  capacity  to  perform  work.  This  is  found 
in  all  living  units.  Work  is  going  on.  Whether  this  energy  is  desig- 
nated as  mechanical,  thermal,  electrical,  chemical  makes  no  particular 
differences  for  the  form  is  only  one  aspect  of  it. 

Energy  may  be  transformed  from  thermal  into  electrical  or  chemical 
into  thermal;  in  fact  "all  forms  of  energy  are  convertible.  The  total 
energy  of  any  substance  or  system  cannot  be  altered  by  the  mutual 
actions  of  its  parts."  Furthermore,  energy  is  practically  indestructible. 

*  Prepared  by  Charles  E.  Marshall  and  Arao  Itano. 

155 


156  PHYSIOLOGY   OF   MICROORGANISMS 

This  is  true  whether  energy  exist  as  potential  energy  in  the  form  of 
rest  or  as  kinetic  energy  in  the  active  form.  "In  every  modification 
of  a  material  system,  not  affected  by  forces  foreign  to  a  system,  the 
sum  of  its  potential  and  kinetic  energies  remains  constant."  This 
statement  represents  the  great  law  of  Conservation  of  Energy.  To 
determine  the  total  energy  of  a  body  is  difficult  for  it  usually  contains 
potential  and  kinetic  energy  which  may  not  find  expression  in  measur- 
able units.  It  follows  that  "the  total  intrinsic  energy  of  a  body  or 
system  of  bodies  is  never  known.  When  bodies  mutually  react  it  is 
only  the  difference  of  the  energy  of  each  body  in  two  states  which  is 
considered.  If  a  body  has  less  energy  in  its  actual  than  in  its  standard 
state  the  expression  for  its  energy  is  negative."  The  functionings 
of  a  living  organism  may  well  be  said  to  be  the  energizing  powers  of 
such  organism.  These  powers  are  maintained  by  the  food  consumed, 
part  of  which  enters  into  the  synthesis  of  new  protoplasm  or  in  renewal 
processes,  and  part  furnishes  the  energy  for  the  work  performed. 
Whether  the  energy  goes  to  constructive  and  reconstructive  purposes  in 
the  maintenance  of  the  mechanism  or  whether  to  the  manifest  liberation 
of  energy  as  work,  it  is  desirable  to  remember  that  the  ramifications  of 
either  route  are  multitudinous,  intricate  and  mostly  concealed.  The 
principle  of  the  Conservation  of  Living  Forces  states  "that  the  differ- 
ence of  the  force-functions  (or  work)  at  the  beginning  and  at  the  end 
of  the  motion  of  a  system  is  equal  to  the  difference  of  the  vires  viva 
(kinetic  energies)  at  the  beginning  and  the  end  of  the  motion."  This 
is  readily  seen  to  represent  only  measurable  and  definite  performance 
and  does  not  in  any  sense  represent  potential  possibilities. 

If  living  energy  is  functional  energy  or  functioning  then  an  under- 
standing of  it  with  any  degree  of  comprehensiveness  means  that  it  is 
to  be  found  in  the  study  of  the  many  functional  processes  of  the  body 
in  detail.  To  this  attention  is  directed. 

SOLUTIONS 

When  any  substance  is  placed  in  or  is  associated  with  another 
substance  in  such  a  manner  as  to  enable  both  to  establish  a  uniform 
ionic,  molecular  and  particulate  mixture,  such  a  combination  is  called 
a  solution.  In  a  true  solution,  this  combination,  in  which  the  ionic  or 
molecular  components  are  intimately  merged  or  blended  and  are 


PHYSICAL  FORCES  INVOLVED  IN  BIOLOGICAL  ACTIVITIES     157 

uniformly  distributed  or  dispersed,  is  considered  a  homogeneous  system. 
It  is  also  a  one-phase  system  because  its  components  are  not  mechanically 
separable  or  physically  different.  On  the  other  hand,  colloidal  solutions 
(see  colloids  and  crystalloids)  consisting  of  components  which  are 
physically  different  and  mechanically  separable  form  a  heterogeneous 
system.  Such  a  system  or  solution  is  therefore  either  diphasic  or 
polyphasic. 

A  true  solution,  composed  of  two  substances,  may  have  one  sub- 
stance regarded  as  that  dissolved  and  the  other  as  the  dissolving  agent. 
If  table  salt  is  dissolved  in  water,  the  salt  would  be  regarded  as  the 
substance  dissolved  in  water  as  the  dissolving  agent.  In  this  case,  salt 
would  be  the  solute  and  water  the  solvent.  However,  just  the  converse 
may  be  equally  true:  water  dissolved  in  salt.  Colloidal  solutions 
are  somewhat  different  because  they  consist  of  fine  or  very  small  par- 
ticles in  suspension  or  emulsion.  In  such  solutions,  therefore,  the 
suspended  particles  in  solution  (called  suspensoids)  or  emulsified 
particles  in  solution  (called  emulsoids)  are  the  substances  distributed  or 
dispersed  in  a  medium  or  menstruum.  The  particles  are  called  the 
disperse  phase  and  the  medium  or  menstruum  the  dispersion  means. 
Together  they  constitute  a  heterogeneous  system,  dispersoids,  also  a 
polyphasic  system.  Some  colloids  approximate  very  closely  ionic  and 
molecular  solution. 

A  diagram  (Fig.  100)  will  aid  in  understanding. 

Solutions 

r  i 

True  Colloidal 


Ionic  Ionic    Purely  colloidal     Suspensions 

and  molecular  and  not  easy        and  emulsions 

molecular          of  easily 

• ' 1  mechanical    '    separated 

separation      mechanically 
Gaseous  Liquid  Solid 


I  I 

Gaseous  Liquid  Solid 


FIG.  100. 


158*  PHYSIOLOGY   OF   MICROORGANISMS 

From  the  diagram  it  wilf  also  be  noted  that,  whether  in  true  solution  or 
colloidal  solution,  solutions  are  not  confined  to  the  action  of  water  on 
some  salt  but  extend  in  like  manner  to  gaseous,  liquid  and  solid  alike, 
as  gas  in  gas,  solid  in  gas,  gas  in  solid,  liquid  in  gas,  liquid  in  solid,  etc. 

The  solute  may  exist  in  the  form  of  a  substance  which  becomes 
electrically  dissociated  into  its  ions,  as  sodium  chloride  is  dissociated 
into  its  ions,  sodium  and  chlorine,  in  water,  when  it  is  called  an  elec- 
trolyte. The  solute  may  be  resolved  only  into  its  molecules  in  water,  as 
sugar,  when  it  is  called  a  non-electrolyte.  Such  substances  are  desig- 
nated usually  as  crystalloids,  but  this  is  not  uniformly  so.  Again, 
instead  of  using  the  term  solute,  in  its  place  there  is  used  the  term 
disperse  phase.  This  is  made  of  small  particles  greater  in  size,  as  a  rule, 
than  molecules,  and  only  in  suspension.  The  solution  takes  on  the 
differentiated  form  as  represented  by  heterogeneous,  polyphasic,  and 
colloidal  characters.  The  so-called  solid  solution  signifies  that  one 
solid  substance  may  become  distributed  throughout  another  solid 
substance  as  in  the  case  of  "crystals  which  are  uniformly  composed 
by  two  crystalline  substances  which  present  similarity  in  crystalline 
form  as  well  as  of  chemical  composition"  or  as  coloring  matter  in 
mineral  salts. 

The  significance  of  the  possibilities  of  solutions  should  be  grasped 
to  understand  the  changes  taking  place  in  protoplasm  through  meta- 
bolism, egestion  and  ingestion. 

ELECTRICAL  CONDUCTIVITY,  IONIZATION  AND  DISSOCIATION 

When  a  salt  as  sodium  chloride  (NaCl)  is  dissolved  in  water  it 
undergoes  dissociation  or  breaks  up  into  atoms — sodium  (Na)  and 
chlorine  (Cl).  Each  atom  is  made  up  of  electrons  some  of  which 
constitute  the  center  and  are  positively  charged  with  electricity  while 
some  are  on  the  outside  and  are  negatively  charged  with  electricity. 
These  latter  are  more  mobile,  are  not  held  so  tenaciously,  and  are  called 
the  -valence  electrons  because  they  establish  the  valency  of  the  atom. 
These  electrons  could  be  regarded  as  the  units  of  electric  charge.  If 
sodium  (Na)  and  chlorine  (Cl)  unite  in  the  formation  of  sodium  chloride 
(NaCl)  it  appears  to  be  by  electric  attraction.  This  may  be  due  to  the 
transfer  of  valence  electrons  from  one  to  the  other  or  attributable 
to  the  rearrangement  of  valence  electrons  within  the  atom  thus  creating 


PHYSICAL  FORCES  INVOLVED  IN  BIOLOGICAL  ACTIVITIES      159 

the  attraction.  The  two  atoms,  however,  remain  electrically  neutral. 
A  ny  free  atom  or  combination  of  atoms  which  carries  a  charge  of  electricity 
is  called  an  ion.  When  sodium  chloride  (NaCl)  is  resolved  into  the  two 
atoms  sodium  (Na)  and  chlorine  (Cl)  in  solution  and  each  contains  an 
electric  charge,  the  process  is  called  ionization  or  electric  dissociation. 
The  sodium  chloride  (NaCl)  or  like  substance  is  called  an  electrolyte. 
The  positive  ions  are  known  as  cations  and  the  negative  as  anions. 


Cathode 


FIG.  101. — Movement  of  Electric  Current  and  Ionization. 

In  the  dissociation  of  sodium  chloride  (NaCl)  in  water  a  negative 
lit  of  electricity  in  the  form  of  a  valence  electron  goes  with  the  chlorine 
(Cl)  and  a  positive  unit  of  electricity  in  the  form  of  a  valence  electron 
is  thus  established  with  the  sodium  (Na).  These  electrons  are  further 
attached  to  molecules  of  water  together  with  which  are  formed  the 
electrically  charged  ions.  The  loss  of  the  negative  valence  electron 
by  the  sodium  (Na)  when  it  passes  with  the  chlorine  atom  (Cl)  leaves 
the  sodium  atom  (Na)  positive  or,  in  other  words,  the  withdrawal  of  the 
negative  electron  from  the  sodium  (Na)  disestablishes  the  neutrality 
of  the  sodium  (Na)  by  making  it  positive  and  that  of  the  chlorine  (Cl) 
by  making  it  negative.  The  result  creates  a  flow  of  electricity  because 
there  is  a  difference  in  the  potential  of  the  two  atoms.  The  potential 
determines  the  flow  of  electricity  in  much  the  same  manner  as  pressure 
determines  the  flow  of  a  liquid. 

Such  resistance  as  is  offered  by  a  solution  to  a  current  of  electricity 
may  be  measured  by  the  Wheatstone  Bridge. 


160  PHYSIOLOGY   OF   MICROORGANISMS 

When  an  electric  current  is  introduced  into  a  solution  of  sodium 
chloride  (NaCl),  the  ions  carry  the  electricity,  sodium  (Na)  carrying 
the  positive  charge  and  chlorine  (Cl)  the  negative  charge.  The  positive 
or  sodium  ions  gather  at  the  electrode  or  pole  or  cathode,  the  pole  by 
which  the  current  leaves  the  solution;  the  chlorine  ions  which  have  the 
negative  charge  pass  against  the  current  to  the  anode,  the  pole  by  which 
the  current  enters  the  solution.  Those  gathered  at  the  cathode  are 
the  cations,  those  at  the  anode,  the  anions. 

The  electric  relations  existing  between  sodium  (Na)  and  chlorine 
(Cl)  in  sodium  chloride  (NaCl)  also  exist  in  hydrochloric  acid  (HC1) 
in  which  hydrogen  and  chlorine  bear  the  sa-me  relationships  as  sodium 
(Na)  and  chlorine  (Cl)  in  sodium  chloride  (NaCl). 

The  strength  of  the  acid  is  measured  by  displaceable  hydrogen  atoms. 
In  nitric  acid  (HNOs)  one  hydrogen  atom  (H)  is  displaceable,  in  acetic 
acid  (CH3COOH)  only  one  hydrogen  atom  (H)  is  displaceable,  for  the 
hydrogen  atoms  of  the  methyl  group  (CH3)  are  not  displaceable. 

If  an  acid  is  neutralized  it  is  the  hydrogen  which  has  been  substi- 
tuted by  some  other  cation. 

Since  it  is  the  hydrogen  (H)  which  determines  the  strength  of  an  acid 
and  this  hydrogen  (H)  has  definite  values  in  every  acid  then  it  must  be 
possible  to  establish  a  standard  of  measurement  by  taking  a  gram- 
molecular  solution  as  of  hydrochloric  acid  (HC1)  which  is  called  a  normal 
(N)  solution.  On  the  other  hand,  alkali  solutions  may  be  established 
of  standard  values  against  the  acid  standards.  In  these  the  hydroxyl 
(OH)  ions  act  as  the  neutralizing  agents  against  hydrogen  (H)  ions 
of  the  acids  in  such  proportion  as  to  form  water. 

"True  Reaction"  ("True  Acidity,"  "True  Alkalinity,"  "True 
Neutrality").* — The  "true  acidity"  of  an  acid  solution  is  brought  about 
by  the  dissociated  (hydrogen)  ions;  therefore  the  acidity  is  proportional 
to  the  concentration  of  the  dissociated  hydrogen  ions,  and  not  to  the 
total  gram  molecules  of  acid  present.  For  example,  if  one-tenth  normal 
hydrochloric  acid  is  taken,  approximately  only  91  per  cent,  of  the  total 
amount  of  acid  becomes  dissociated.  The  "true  acidity,"  i.e.,  the 
hydrogen  ion  concentration,  of  this  solution  is  only  91  per  cent,  of  the 
one-tenth  normal  hydrochloric  acid,  or  ninety-one  thousandths  normal. 
The  dissociation  of  weak  acid  is  still  less.  For  instance,  in  a  solution  of 

*  Bull.  167,  Mass.  Agr.  Exp.  Sta.  by  Arao  Itano. 


PHYSICAL  FORCES   INVOLVED   IN  BIOLOGICAL  ACTIVITIES      l6l 

one-  tenth  normal  acetic  acid  only  i  .  3  per  cent,  approximately  of  the 
total  acid  is  dissociated,  and  the  hydrogen  ion  concentration  of  this 
solution  is  therefore  thirteen  ten-thousandths  normal.  The  "true 
acidity"  of  one-  tenth  normal  hydrochloric  acid  is  also  about  seventy 
times  greater  than  that  of  one-tenth  normal  acetic  acid,  although  both 
solutions  contain  the  same  amount  of  acid. 

The  same  holds  true  with  the  electrically  dissociated  base  in  which 
the  metallic  and  hydroxyl  ions  are  dissociated.  The  "true  alkalinity" 
of  such  a  solution  is  not  determined  by  the  total  amount  of  base 
present,  but  exclusively  by  the  concentration  of  dissociated  hydroxyl 
ions.  For  example,  in  a  one-tenth  normal  solution  of  the  strong  base, 
sodium  hydroxide,  about  84  per  cent,  of  the  total  amount  of  the  base  is 
dissociated,  and  in  the  case  of  a  weak  base,  such  as  ammonium  hy- 
droxide, approximately  1.4  per  cent,  of  the  total  amount  of  the  base. 
The  "true  alkalinity"  of  these  solutions,  therefore,  is  eighty-four 
thousandths  normal  and  fourteen  thousandths  normal,  respectively. 
Thus,  regarding  the  alkalinity  as  in  the  case  of  acidity,  we  may  say  in 
conclusion  that  "true  alkalinity"  of  a  solution  is  proportional  to  the 
concentration  of  hydroxyl  ions. 

From  the  above  discussion,  "true  neutrality"  of  a  solution  may  be 
stated  as  follows  :  it  is  a  solution  in  which  the  same  amount  of  H  and  OH 
ions  are  present.  For  example,  a  "true  neutral  solution,"  viz.,  pure 
water,  contains  as  many  hydrogen  ions  as  hydroxyl  ions.  It  can  be 
expressed  as  follows:  — 

+ 

H2O±=»H+OH 

_L          _  . 

in  which  CTT=  CTT,  ^  indicating  the  concentration. 


Again,  a  solution  may  not  necessarily  be  neutral,  although  it  con- 
tains equivalent  quantities  of  acid  and  alkali.  For  example,  if  a 
solution  which  contains  hydrochloric  acid  and  sodium  hydroxide  is 
taken,  it  can  be  expressed  in  the  following  manner:  — 

+         -  +  +  - 

H        Cl     +     Na  OH       =  Na  Cl  +  HOH 

hydrochloric  sodium  hydroxide  salt  water 

acid 

This  solution  is  neutral  only  when  it  contains  just  as  many  hydrogen 
as   hydroxyl   ions,   or   when   both   the   acid  and    alkali  are  equally 
dissociated. 
11 


1  62  PHYSIOLOGY   OF   MICROORGANISMS 

It  is  understood,  therefore,  that  the  "true  acidity,  alkalinity  and 
neutrality"  are  not  determined  by  the  amount  of  such  substances 
present,  but  entirely  by  the  H  and  OH  ion  concentration. 

Theory  of  H  Ion  Concentration.  —  The  announcement  of  the  theory  of 
electric  dissociation  by  Svante  Arrhenius,  in  1887,  marked  a  new  era  in 
physical  chemistry.  It  was  F.  Kohlrausch  and  A.  Heydweiller  who 
demonstrated  that  even  the  purest  water  is  a  conductor  of  electricity,  and 
accordingly  prepared  a  distilled  water  of  the  least  specific  conductance. 
They  measured  the  specific  conductance  by  means  of  electric  conduc- 
tivity. Later,  other  methods  for  the  estimation  of  dissociation  were 
established,  and  the  results  obtained  by  Kohlrausch  were  confirmed. 
Now  it  is  proved  that  a  very  small  portion  of  the  water  molecule  is 
dissociated  into  two  electrically  charged  parts  (or  ions),  as  follows: 


H2O  <=>  H  +  OH 

Its  dissociation  takes  place  according  to  the  law  of  mass  action  in 
accordance  with  the  following  equation  :  — 

(H)(OH) 
~050T 

in  which  K  denotes  the  ionization  constant;  that  is  to  say,  the  product  of 
the  hydrogen  and  hydroxyl  ion  concentration,  divided  by  the  concentra- 
tion of  the  undissociated  water  molecule,  should  be  constant. 

The  concentration  of  water  is  generally  constant.     Therefore  it  may 
be  expressed  as  follows:  — 

(H).(OH)  =  Kw  (2) 

in  which  Kw  denoted  K.H2O,  or  ionization  constant  of  water. 

Equation  (2)  is  another  form  of  equation  (i). 

This  ionization  constant  of  water  has  been  determined  by  several 
noted  physical  chemists,  and  found  to  be  io~14  at  22°;  that  is, 

NOTE.  —  (H)  and  (OH)  express  the  concentration. 

(H).(OH)  =  Kw      or 

Kw  =  iQ~14  (3) 


PHYSICAL  FORCES  INVOLVED  IN  BIOLOGICAL  ACTIVITIES      163 

Since  pure  water  is  a  neutral  solution  it  contains  the  same  number  of 
dissociated  hydrogen  and  hydroxyl  ions.  Therefore  equation  (3)  can  be 
expressed  as  follows: — 

io-7  X  io~7  =  io-14          (4) 

That  is,  a  pure  water  contains  of  each  io~7  dissociated  hydrogen  and 
hydroxyl  ions,  or  .000000 1  gram  ions  per  litre,  which  is,  in  a  general 

N 

term,  one  ten-millionth  normal  -  —     The  acidity,  alkalinity 

10,000,000 

and  neutrality,  therefore,  are  expressed  in  terms  of  hydrogen  ion 
concentration  in  the  following  manner: — 

Acid  reaction  (H)  >  io~7 
Alkaline  reaction  (H)  <  io~7 
Neutral  reaction  (H)  =  io~7 

That  is,  in  an  acid  solution  there  are  more  than  -  —gram 

10,000,000 

molecule  of  dissociated  hydrogen;  in  an  alkaline  solution,  less;  and  in  a 

neutral  solution,  just  -  gram  molecule.     Thus  the  reaction  is 

10,000,000  ° 

usually  expressed  in  terms  of  hydrogen  ion  concentration  unless  it  is 
indicated  otherwise. 

From  the  above  discussions  it  is  readily  seen  that  if  the  ionization 
constant  is  known,  and  the  hydrogen  ion  concentration  is  determined 
experimentally,  then  the  hydroxyl  ion  concentration  can  be  calculated. 
The  determination  of  hydrogen  ion  concentration  is  accomplished  by 
the  use  of  the  gas  cell,  of  which  the  principle  is  based  upon  the  potential 
of  the  chain.  This  chain  as  described  in  physical  chemistry,  consists 
of— 

Hg-HgCl  |  n/ioKCl  |  cone.  KC1  |  solution  |       Ft  H2 

calomel  electrode  concentrate  (unknown)         platinum  elec- 

potassium  trode  saturated 

chloride  with  hydrogen 

in  a  dish.  gas. 

The  potential  of  such  a  chain  can  be  determined  by  the  usual  physical 
method.  Then  the  relation  between  the  measurement  of  potential  and 
hydrogen  ion  concentration  can  be  calculated  by  the  following  equation  :— 

Pa  P  ~  °-3377 

0.0577  +  0.0002  (t°  -  18°) 

NOTE. — (X)  =  notation  of  the  concentration  of  ions. 


164  PHYSIOLOGY   OF   MICROORGANISMS 

where — 

PH — the  term  adopted  by  S.  P.  L.  Sorensen  to  express  the  exponent 

of  gm.-  equivalent  of  hydrogen  ions  per  liter. 
P — the  total  E.  M.  F.  of  the  chain.     It  can  be  determined  by  the 
following  equation,  having  the  apparatus  arranged  as  it  is 
shown  in  the  diagram: — 

P  =  -  '  -     -  in  which  RI — the  bridge  reading  for  the  chain  against 

an  accumulator. 

R  — the   bridge   reading  for   the   ac- 
cumulator against  the  normal  ele- 
ment. 
1.0189 — the  voltage  of  the  normal  element  at 

1 8°  (standard). 

0.3377 — the  sum  of  potential  of  calomel  electrode  (N/io  KC1)  and 
hydrogen  electrode  in  a  solution  where  the  hydrogen 
concentration  is  normal  (H)  =  i  or  PH  =  o. 

0.0577 — thermodynamical  factor  at  18°  which  is  influenced  by  tem- 
perature, 0.0002  for  each  degree  centigrade,  or  it  changes 
as  follows : — 

0.0577  +  0.0002  (t°  —  1 8°),  of  which  t°  equals  temperature 
at  the  time  of  determination. 

After  PH  is  determined  it  is  necessary  to  understand  the  value  of 
H-ion  concentration,  although  the  experimental  results  are  generally 
expressed  in  PH.  It  will  be  shown  at  the  end  of  an  example,  illustrating 
the  application  of  the  formula  as  well. 


Example. 

t°  =  i9.2°C  (constant  during  the  experiment). 
RI  =  307.0   (constant  reading  on   the  bridge  at  five  minute 

interval). 

R    =  500.2.  (as  above). 
M.  F.  of  the  normal  element  =  1.0189. 


PHYSICAL  FORCES  INVOLVED  IN  BIOLOGICAL  ACTIVITIES      165 

Then  the  total  E.  M.  F.  of  the  chain  can  be  calculated  as  follows: — 

307.0  _     x        500.2  =  N.E.  N.E.=  normal  element. 

1000       Ac.'      1000        Ac.  Ac.  =  accumulator. 
_  307.0 _Ac.            ,  ,  x  =  the  chain. 

1000  1000  =  scale  on  bridge. 

500.2  _  N.E. 
1000         Ac. 

1000  N.E. 
Ac.  = 

500.2 

1000  X  1.0189  ,  , 

~^oT~ 

Substituting  (2)  in  (i), 

_  3°7-°  \/  I000  X  1-0189 
x  —  /\ 

1000  500.2 

=  0.6254  volt,  which  is  expressed  p. 

Substituting  the  value  for  p  in  the  formula, 

0.525-0.3377 

•EH    — 


0.0577  +  0.0002  (19.2  —  18) 

=   4.967 

or  in  terms  of  H  ion  concentration, 

PH  =  4-967  =  -  4-967(1°g-  H) 
IO-4-967  =  1>07g9  x  IO-5 

H  =  0.000010789 

Besides  the  apparatus  listed  below,  a  H-generator  was  employed, 
which  is  a  good-sized  Kipp's  generator  used  with  a  series  of  washing 
bottles  and  drying  tube,  consisting  of  (a)  30  per  cent.  KOH,  (b)  alkaline 
pyrogallic  acid,  (c)  cone .  H2SO4  and  soda  lime  in  U-tube.  Since  a  consid- 
erable amount  of  CO2  is  produced  during  the  course  of  metabolism,  the 
same  precaution  is  taken  as  with  blood.  For  this  purpose  Hasselbach's 
electrode  with  shaking  arrangement  is  employed. 

In  setting  up  the  apparatus  special  attention  should  be  paid  to 
rigidness,  insulation  and  temperature.  In  order  to  meet  with  these 
requisites  the  apparatus  was  placed  on  a  big  central  table  in  the  labora- 
tory. First,  one  dozen  large  glass  rings  of  the  same  height  were 


1 66 


PHYSIOLOGY   OF   MICROORGANISMS 


distributed  over  the  top  of  the  table.  These  supported  a  thick  glass 
plate  on  which  several  blocks  of  paraffine  for  each  piece  of  apparatus 
were  placed.  Thus  it  was  possible  to  obtain  a  perfect  insulation. 

In  preparing  the  different  parts  of  the  apparatus  extreme  care  should 
be  exercised  to  obtain  an  accurate  result.  The  method  for  the  prepara- 
tion of  the  normal  element,  calomel  electrode,  gas  cell,  and  also  calibra~ 
tion  of  the  bridge  wire,  etc.,  is  described  in  detail  in  Findlay's  "  Practical 


jFio.  102. — Apparatus  employed  in  determination  of  H-ion  concentration. 
DESCRIPTION  OF  DIAGRAM 


LI — Lippmann's  capillarimeter. 

L2 — Tungsten  lamp. 

A  — Accumulator. 

N — Western  normal  element. 

Si — Switch  with  quick  short  circuiting  key. 

82 — Three-way  switch. 


S3 — Two-way  switch. 

C  — Calomel  electrode. 

K  —Concentrated  KC1  cup. 

G—  Gas  cell. 

B — Bridge. 

P— Thick  glass  plate. 


Physical  Chemistry."  Every  contact  should  be  carefully  made,  so  that 
accurate  readings  can  be  obtained.  It  is  worthy  of  mention  that  the 
diffusion  potential  between  n/io  KC1  calomel  electrode  and  the  solution 
to  be  tested  is  reduced  by  interposing  the  saturated  solution  of  KC1  as  it 
is  indicated  by  K  on  the  diagram.  For  the  standardization  of  the  elec- 
trode it  was  first  platinized  with  general  precaution;  then  the  hydrogen 
ion  concentration  of  the  mixed  solution  (7  c.c.  of  m/i5  KH^PO^  3  c.c.  of 


PHYSICAL  FORCES  INVOLVED  IN  BIOLOGICAL  ACTIVITIES      167 

m/ 1 5  Na2HPO4)  was  determined  at  different  intervals.  After  the  read- 
ings became  constant  there  was  a  difference  of  0.0005  volts  between  the 
theoretical  data  and  the  results  obtained. 

With  the  above  facts  in  mind  it  becomes  possible  to  enter  upon  a 
more  intelligent  discussion  of  the  methods  involved.  It  has  been  stated 
previously  that  most  microbiological  experiments,  having  for  their 
purpose  the  study  of  reaction  upon  microbial  life,  fall  under  the  follow- 
ing procedures: — 

(a)  Kisch's  method. 

(b)  Ordinary  titration  method. 

(c)  Colorimetric  method. 

It  is  well  known  that  Kisch's  method  is  a  dilution  method  wherein  a 
certain  number  of  gram  molecules  of  an 'acid  or  alkali  are  diluted  to  a  defi- 
nite quantity  for  the  purpose  of  ascertaining  the  influence  of  the  reac- 
tion upon  the  life  of  bacteria.  There  are  two  distinct  ways  to  apply 
Kisch's  method,  namely:  (a)  immersing  the  bacteria  in  different  dilu- 
tions of  acids  or  alkalis  in  pure  water  for  different  periods  of  time  by 
means  of  silk  threads  or  any  other  convenient  agents,  and  then  testing 
their  vitality;  or  (b)  adding  a  known  percentage  of  acids  or  alkalis 
directly  to  the  culture  medium  (usually  solution).  In  either  case  the 
results  obtained  by  Kisch's  method  indicate  neither  the  influence  of 
"true  reaction"  upon  microbial  life  nor  the  influence  of  molecular 
concentration,  because,  as  Lingelheim  has  shown,  different  acids  of 
the  same  molecular  concentration  have  varying  influence  upon  micro- 
organisms, and  the  degree  of  influence  is  parallel  to  the  dissociation 
constant  of  an  acid  or  alkali.  This  is  especially  true  in  the  case  of 
the  second  manner  of  application,  (b) ,  where  adsorption  is  caused  by  the 
culture  medium. 

The  ordinary  titration  method  is  generally  employed  in  adjusting 
reaction  of  culture  medium,  and  also  to  measure  the  amount  of  acid  or 
alkali  produced  in  the  course  of  physiological  tests.  This  method  is 
inaccurate  in  the  study  of  physiological  liquids  containing  more  or  less 
amphoteric  substances  and  a  comparatively  small  quantity  of  H  or  OH 
ions.  In  other  words,  it  is  impossible  to  determine  the  "  true  reaction" 
in  such  a  liquid  by  this  method.  Fuller's  and  Schultz's  methods  of 
adjusting  the  scale  of  reaction  of  culture  media  are  scientifically  con- 
demned by  the  recent  investigation  of  Clark,  who  showed  the  fallacies 
of  the  titrimetric  method.  Again,  the  adsorption  phenomenon  caused 


1 68  PHYSIOLOGY   OF   MICROORGANISMS 

by  the  amphoteric  substance  in  the  course  of  titration  is  well  known, 
and,  in  the  case  of  albumin,  is  usually  expressed  in  the  following 
manner: — 

i  

In  acid  solution    H.  albumin.  OH  =  H  albumin  +  OH 

+ 
In  alkali  solution  H.  albumin.  OH  =  Albumin  OH  +  H 

The  correctness  of  the  above  statement  has  been  experimentally 
demonstrated  by  Sorensen,  Clark  and  others. 

In  many  cases  the  colorimetric  method  gives  fairly  accurate  results, 
but  it  has  been  noted  that  the  presence  of  neutral  salts  as  well  as  ampho- 
teric substances  interfere  with  the  determination.  It  may,  however,  be 
employed  successfully  if  it  is  standardized  for  the  particular  liquid. 
Lately  Clark  and Lubs  employed  the  principle  of  the  colorimetric  method 
for  the  differentiation  of  the  colon-aerogenes  family,  using  suitable 
indicators.  They  have  based  their  experiment  upon  the  wide  diver- 
gence of  the  hydrogen  ion  concentration  in  a  culture  of  one  group  and 
of  the  other,  and  distinguished  this  difference  by  means  of  paranitro- 
phenol  or  methyl  red.  The  use  of  this  method  for  physiologic  work 
other  than  for  microbiology  has  been  practiced  by  many.  Sorensen 
and  Palitzsch  determined  the  hydrogen  ion  concentration  of  sea  water. 
Henderson  and  Palmer  used  it  in  determining  the  acidity  of  urine  to 
diagnose  normal  and  abnormal  conditions.  In  any  case,  the  colori- 
metric method  should  be  standardized  previous  to  its  use,  by  means  of 
the  hydrogen  electrode. 

Examining  these  methods  critically  in  the  light  of  physical  chemistry 
they  are  not  satisfactory  for  the  purpose  of  ascertaining  the  influence  of 
the  so-called  "  true-reaction "  upon  microbial  life.  The  hydrogen 
electrode  was  devised  to  determine  the  hydrogen  ion  concentration, 
and  it  has  been  used  successfully  in  biologic  fields. 

SURFACE  TENSION 

Due  to  such  forces  as  cohesion  and  adhesion  the  particles  of  bodies 
have  a  tendency  to  come  together  in  the  same  manner  as  bodies  fall  to 
the  earth.  This  property  appears  to  lie  within  the  molecular  forces 
of  the  body  and  seems  to  have  a  circumscribed  and  limited  area  of 
action.  If  a  center  is  assumed  in  the  form  of  a  molecule,  this  area 


PHYSICAL  FORCES  INVOLVED  IN  BIOLOGICAL  ACTIVITIES      169 

over  which  an  influence  of  attraction  is  exerted  would  be  in  the  form 
of  a  sphere  and  would  be  recognized  as  the  sphere  of  molecular  action. 
The  layer  of  a  liquid  representing  its  surface  plane  with  a  depth 
equal  to  the  radius  of  the  sphere  of  molecular  action  would  be  the  surface 
film.  If  a  particle  lies  within  or  inside  of  this  surface  film  it  follows 
that  with  this  particle  as  a  center,  the  radius  of  its  sphere  of  activity 
will  extend  beyond  and  above  the  surface  film,  but  if  this  particle  lies 
without  and  below  this  surface  film  the  molecular  forces  on  all  sides  will 
be  equal  and  an  equilibrium  established. 


FIG.  103. — Illustrating  surface  forces. 

This  is  illustrated  in  Fig.  103.  AB  is  the  plane  surface  of  a  liquid. 
N  is  a  particle  with  its  circumference  indicated  in  which  all  forces  are 
equalized.  N'  is  a  particle  in  which  the  forces  downward  are  greater 
than  the  forces  upward.  The  forces  lying  above  the  plane  surface  of 
the  liquid  AB  appear  to  be  less  than  the  forces  operating  immediately 
below  the  plane  surface  AB  in  the  liquid,  yielding  a  considerable 
increase  of  pressure  in  the  liquid.  This  increased  pressure  is  known 
as  the  molecular  pressure  of  the  liquid. 

The  surface  film  described  above  possesses  a  pull  or  is  under  tension 
or  is  the  surface  tension  of  the  liquid.  If  an  iron  ring  has  stretched  across 
its  interior  surface  a  soap  film  and  a  silk-thread  loop  is  carefully  rested 
upon  it  and  run  to  the  iron  ring,  the  film  inside  the  silk  loop  may  be 
broken  readily  by  any  penetrating  substance  when  the  sides  of  the  loop 
will  spread  out  in  the  fullest  degree  drawn  by  the  soap  film  without. 
Much  like  this  is  the  floating  of  a  rubber  band  on  water.  If  a  rod 
dipped  into  alcohol  is  touched  to  the  surface  of  the  water  within  the 
band  the  water  film  without  pulls  the  band  into  its  full  circular  form 
(Fig.  1046)  through  the  reduction  of  the  surface  tension  of  the  water 


170  PHYSIOLOGY   OF   MICROORGANISMS 

within  by  the  addition  of  alcohol.  This  pull  of  the  water  without  may 
be  broken  by  the  addition  of  a  trace  of  alcohol.  In  this  case  the  rubber 
band  again  resumes  its  former  shape  (Fig.  1040). 


L 

FIG.  104. — Illustrating  surface  pull. 


In  the  case  of  an  oil  drop  on  water  the  oil  runs  to  a  ball  because  of 
the  cohesive  forces  within  the  oil  and  the  lack  of  sufficient  gravitational 
and  molecular  forces  or  pulling  forces  within  the  water  film.  Mercury 
for  the  same  reason  distributes  itself  in  many  small  globules  when  split. 
On  the  other  hand  if  the  forces  below  or  upward  attraction  has  a 
stronger  pull  than  the  cohesive  forces,  then  the  oil  would  spread  out  as 
on  a  clean  glass. 

The  definite  reactions  resulting  from  experiments  as  employed  in 
demonstrations  of  the  above  nature  at  once  establish  the  possibility 
of  accurate  quantitative  measurements.  It  has  been  found  that 
substances  vary  very  materially  in  their  surface  tensions.  Kimball* 
gives  the  following  table: — 

SURFACE  TENSIONS  IN  DYNES  PER  CENTIMETER 

Air  Water  Mercury 

Water 73-5              412 

Mercury 539-°  412 

Olive  oil 34-3  20.6                335 

Alcohol 24 . 5               

Ether 17 -6 

*  "College  Physics."     For  Method  of  Measurement,  also  consult  Kimball. 


PHYSICAL  FORCES   INVOLVED   IN  BIOLOGICAL  ACTIVITIES      Ijl 

The  possible  effect  surface  tension  may  have  upon  the  outer  layer  of 
protoplasm  constituting  a  cell  and  in  the  formation  of  a  membrane, 
its  relation  to  nutritional  functioning  and  in  cellular  movements,  its 
suggestiveness  in  connection  with  form  and  its  probable  importance 
with  alterations  of  various  kinds  render  it  a  topic  of  prime  import- 
ance although  its  values  are  very  much  dimmed  by  incomplete 
knowledge. 

ADSORPTION 

Spongy  platinum  has  the  power  to  take  up  considerable  quantities 
of  hydrogen  gas  and  also  oxygen  gas  into  its  mass;  charcoal  takes  color- 
ing material  from  solutions;  it  also  takes  up  gases;  platinum  black  takes 
up  acetic  acid;  calcium  carbonate  takes  up  sodium  nitrate.  When 
substances  are  so  taken  they  are  said  to  be  adsorbed.  This  power  seems 
to  be  resident  in  the  adhesive  forces  of  the  extensive  surfaces  which 
exist  through  the  multiplicity  of  particles  in  the  substance  as  in  char  coal. 
It  has  been  defined  as  the  local  concentration  or  condensation  of  dis- 
solved substances  at  the  interface  between  two  phases.  For  instance, 
the  interface  existing  between  the  dispersoid  phase  and  dispersion 
means  intensifies  the  surface  action  to  such  an  extent  that  there  is  a 
concentration,  a  condensation.  Reactions  are  apparently  accelerated. 
The  contact  of  hydrogen  and  oxygen  in  spongy  platinum  produces 
water.  The  action  many  times  is  that  of  catalysis  as  the  oxidation  of 
alcohol  to  acetic  acid  by  platinum  black.  The  adsorbing  substance 
does  not  seem  to  enter  into  the  chemical  reaction  which  may  occur  but 
may  be  recovered  intact. 

These  reactions  are  influenced  by  temperature,  pressure,  electric 
forces  and  nature  of  the  substance. 

By  this  phenomenon  of  nature  soluble  salts  are  held  back  in  soils 
and  not  washed  away  by  rains.  The  action  of  certain  disinfectants  is 
explained  by  the  deposition  or  concentration  on  the  surfaces  of  micro- 
organisms; the  reaction  of  toxin  with  antitoxin  simulates  adsorption 
phenomena  more  closely  than  mass  action;  the  sensitization  of  bacteria 
by  opsonins  and  the  ingestion  by  leucocytes  also  resemble  adsorption 
acts;  the  peculiar  reactions  of  enzymes  are  regarded  as  similar  to  ad- 
sorption; the  formation  of  a  membrane  upon  exposed  protoplasm  in 
the  case  of  a  crushed  protozoon  also  appears  to  be  the  result  of  the 
adsorptive  action  of  certain  substances. 


172 


PHYSIOLOGY   OF   MICROORGANISMS 

BROWNIAN  MOTION 


This  phenomenon  is  familiar  to  students  of  microbiology.  When 
studying  some  bacteria  in  a  hanging-drop  under  one-twelfth  oil  immer- 
sion objective,  this  movement  may  be  seen.  It  is  not  only  visible  with 
some  of  these  living  organisms  but  extends  to  many  substances  existing 
in  very  fine  particles  and  suspended  in  certain  media.  It  is  a  common 
phenomenon  among  colloidal  solutions. 


FIG.  105. — Illustrating  Brownian  movement  (After  Perrin). 

The  character  of  the  movement  is  well  illustrated  by  Perrin*  (Fig. 
105)  who  has  made  a  special  study  of  the  subject.  The  path  is  a 
straight  line  until  opposed  when  it  rebounds  in  another  straight  line 
producing  a  zig-zag  route. 

The  cause  of  the  motion  appears  to  be  inherent  in  the  molecular 
movements  of  the  dispersion  means  of  a  colloid,  of  the  liquid  in  which 
the  particles  are  suspended.  The  direction  of  the  particles  as  stated 
above,  is  that  of  a  straight  line  until  a  collision  with  the  invisible  mole- 
cules takes  place  when  the  rebound  sends  the  .particles  in  a  straight  line 
in  another  direction.  This  process  continues  indefinitely.  The 

*  Perrin,  M.  Jean,  Brownian  Movement  and  Molecular  Reality. 


PHYSICAL  FORCES  INVOLVED  IN  BIOLOGICAL  ACTIVITIES      173 

particle  subject  to  these  molecular  movements  and  forces  responds  on 
the  whole  as  a  football  might  be  knocked  indiscriminately  about  a  field 
by  a  group  of  unorganized  school-boys. 

Such  movements  of  colloidal  particles  are  supposed  to  render 
colloidal  solutions  more  stable.  This  taken  together  with  the  density 
of  the  dispersion  means,  its  viscosity,  the  size  of  the  particles  in  which 
surface  action  becomes  more  evident,  and  the  electric  charge  probably 
accounts  in  large  part  for  the  permanency  of  the  dispersoid  state.  The 
velocity  of  the  movement  of  particles  depends  upon  many  of  the  factors 
associated  with  colloidal  permanency.  An  increase  of  temperature 
quickens  the  movement  not  through  convection  currents  but  by  the 
molecular  activity;  viscosity  acts  in  a  seeming  frictional  capacity; 
the  density  acts  as  if  there  was  a  tendency  to  close  in  on  the  particles 
with  forces  which  are  made  effective  through  the  multiplicity  of  mole- 
cules; size  apparently  is  much  like  keeping  a  small  ball  in  the  air  as 
compared  with  a  large  ball. 

Again,  the  size  of  particles  which  are  subject  to  exact  measurement 
is  Delated  to  the  rapidity  of  their  movement.  Exner  has  made  this 
comparison : — 

Diameter  of  par-  Velocity  of  particle 

ticle  in  ft  in  M  per  second 

1.3  2.7 

0-9  3-3 

0.4  3.8 

It  will  be  seen  that  the  smaller  they  are  the  more  rapidly  they  move. 

Brownian  motion,  because  of  the  forceful  drive  furnished  by  the 
molecules,  appears  to  be  an  important  factor  in  diffusion  and  osmotic 
bearings. 

DIFFUSION,  OSMOSIS,  DIALYSIS,  PERMEABILITY 

If  a  twelve  per  cent,  warm  gelatin  solution  is  brought  in  contact 
with  water  of  the  same  temperature,  currents,  not  convection  currents, 
are  seen  radiating,  spreading  and  extending  from  the  gelatin  solution 
into  the  water  until  finally  they  merge  with  the  water  and  are  lost  to 
sight,  when  the  entire  mass  becomes  uniform  and  homogeneous.  A 
strong  salt  solution,  when  placed  in  the  bottom  of  a  cylinder  and  water 
carefully  poured  above  it,  will  little  by  little  work  up  into  the  water 
until  the  whole  is  one  homogeneous  concentration.  This  would  also 


174  PHYSIOLOGY   OF   MICROORGANISMS 

be  true  if  the  water  in  the  former  case  were  substituted  by  a  weaker 
solution  of  gelatin  or,  in  the  latter  case,  by  a  weaker  solution  of  salt. 
There  is  a  tendency  to  equalize  or  become  uniform  and  homogeneous. 

Microbiologists  are  also  familiar  with  certain  special  phenomena. 
Litmus  agar  becomes  reduced  by  the  growth  of  microorganisms.  Oxy- 
gen has  been  consumed.  When  the  culture  is  allowed  to  remain  ex- 
posed to  the  air  for  a  time,  the  microorganisms  cease  to  grow  and 
multiply;  the  litmus,  beginning  at  the  top,  gradually  resumes  its  color 
as  the  air  works  its  way  down  through  the  culture.  There  has  been  a 
gradual  diffusion  of  the  air  throughout  the  litmus  agar.  Many  cul- 
tural phenomena  could  be  recalled  in  this  connection.  One  will  suffice. 
The  heating  of  culture  media  to  drive  off  the  air  for  anaerobic  cultivation 
is  of  frequent  occurrence,  for  it  is  well  known  how  the  air  soon  penetrates 
when  media  are  allowed  to  stand. 

Apparently  there  are  encountered  in  the  first  two  paragraphs  dis- 
tinct phenomena  or  a  single  phenomenon  modified  in  the  one  or  the 
other  instance.  The  usual  explanation,  however,  is  covered  by  the 
word  "diffusion." 

The  recent  developments  in  the  understanding  of  diffusion  attribute 
to  diffusion  the  same  forces  operating  in  gases.  It  is  the  drive  possessed 
by  the  molecules  to  expand  or  press  out  until  equalization  or  equilibrium 
is  established.  This  movement  is  from  the  more  concentrated  solution 
toward  the  less  concentrated  or  toward  the  pure  solvent.  The  nature 
of  a  substance,  difference  in  concentration  and  temperature  materially 
influence  this  movement. 

This  accords  with  the  forces  of  osmosis  as  well:  The  pressure  upon 
the  obstructing  membrane  through  which  the  particles,  molecules  or 
ions  of  a  substance  are  attempting  to  make  their  way  is  called  osmotic 
pressure;  the  particles  are  held  back  or  restrained  in  their  movements 
outward.  It  has  been  found,  however,  that  "  the  osmotic  pressure  of  a 
dissolved  substance  is  exactly  the  same  as  the  gas-pressure  which 
would  be  exerted  if  the  solvent  were  removed  and  the  dissolved  sub- 
stance in  gaseous  form  were  left  behind  to  occupy  the  same  volume 
at  the  same  temperature."  It  is  also  known  that  "where  two  liquids 
which  will  mix  are  separated  only  by  a  porous  membrane  there  is  a 
movement  of  the  liquid  in  both  directions  through  the,  membrane. 
The  greater  movement  is  usually  from  the  less  dense  to  the  more  dense 
so  as  to  cause  the  line  of  the  more  dense  liquid  to  rise  above  that  of  the 


PHYSICAL  FORCES  INVOLVED  IN  BIOLOGICAL  ACTIVITIES      175 

less  dense.  This  action  increases  with  the  temperature  and  is  pro- 
portional to  the  concentration  of  the  solution.  In  the  illustration  of 
diffusion  above  by  means  of  gelatin  and  salt,  diffusion  follows  its  natural 
course;  but  in  the  case  of  oxygen  penetrating  litmus  agar  or  any  other 
medium  the  action  may  be  regarded  as  modified  diffusion  or  osmosis  in 
which  the  medium  acts  as  a  barrier  to  the  medium-content  but  allows 
the  gas  (air)  in  its  drive  onward  to  pass  and  diffuse  throughout.  This 
leads  to  the  significance  of  permeability  of  membranes. 

Much  attention  has  been  given  to  the  study  of  membranes  as 
they  relate  so  closely  to  the  membranes  of  cells  which  are  concerned 
with  living  processes.  It  is  more  or  less  simple  to  demonstrate 
the  passage  of  water  and  the  restraining  of  a  substance  like  sugar 
by  means  of  parchment  paper.  This  is  a  common  experiment.  In  a 
thistle  tube  with  its  mouth  covered  with  parchment  paper  place  a 
sugar  solution  to  the  neck.  When  plunged  into  water,  the  water  will 
pass  in  and  appear  in  the  rising  line.  At  the  same  time  no  sugar 
passes  through  and  out  into  the  water.  The  molecules  appear  too 
large  to  pass  through  the  pores.  This  membrane  is  semi-permeable 
since  it  permits  water  to  pass  but  restrains  sugar.  A  membrane  or 
anything  which  does  not  allow  anything  to  pass,  as  glass,  would  be 
called  impermeable. 

Whether  dialysis  (passage  through  a  membrane  in  the  separation 
of  colloids  and  crystalloids)  or  the  permeability  of  membranes  is 
traceable  to  its  sieve-like  nature,  its  chemical  reaction,  or  to  its  solvent 
action  or  to  more  than  one  of  these  is  a  mooted  problem  of  prime 
interest  but  out  of  place  in  this  consideration.  Some  data  throwing 
light  on  the  action  of  membranes  may  be  helpful,  however.  The 
Bechhold  ultra-filters  made  of  collodion,  which  may  be  graded  to  vary- 
ing porosities,  have  been  employed  in  such  a  manner  as  to  illustrate  the 
permeability  of  membranes.  Some  substances  will  pass  while  others 
will  not  until  the  size  of  pores  are  adjusted.  The  membrane  resulting 
by  the  contact  of  potassium  ferrocyanide  with  copper  sulphate  allows 
water  and  potassium  chloride  to  pass  while  it  withholds  potassium  sul- 
phate and  other'  salts.  In  nature  membranes  may  be  permeable  to 
certain  salts  at  times  and  impermeable  at  other  times.  Osterhout 
has  demonstrated  many  of  the  possibilities  of  protoplasmic  permea- 
bility. Speaking  in  very  general  terms,  permeability  as  manifested  in 
living  cells  and  measured  by  electric  conductivity,  as  has  been  the  case 


1 76  PHYSIOLOGY   OF   MICROORGANISMS 

with  Osterhout's  investigations,  may  be  decreased  in  its  reaction  to 
sodium  chloride  by  alkaloids,  as  caffein,  nicotine  and  cevadine,  by  bile 
salts  as  sodium  taurocholate,  and  by  acids  as  hydrochloric  acid;  on  the 
other  hand,  .it  is  increased  by  alkalis,  by  certain  isotonic  combinations 
of  salts  or  balanced  solutions  and  by  acids  following  the  first  stimula- 
tion. Protoplasm  may  vary  widely  from  the  normal  in  its  permeability 
and  both  vegetable  and  animal  cells  respond  in  much  the  same  general 
manner. 

Although  these  specific  facts  may  be  very  limited  compared  with 
the  entire  field  of  permeability  possibilities  to  which  a  living  organism 
is  exposed,  they  do,  however,  indicate  that  the  membrane  or  protoplas- 
mic protective  surfaces  have  the  power  to  act  in  a  selective  manner 
per  se  or  to  yield  to  environing  forces  or  influences  which  control  or 
make  life  possible  by  antagonisms,  reactions,  neutralizations  and  other 
agencies  among  themselves.* 

Osmotic  pressure,  following  the  laws  of  gas  pressure,  represents 
the  pressure  exerted  by  the  particles  of  a  given  volume  of  a  solution. 
The  particles,  molecules,  or  ions,  of  the  solution,  as  in  gas  are  constantly 
on  an  outward  drive,  an  expansive  drive,  and  they  carry  with  them 
much  force  which  is  proportional  to  the  concentration  of  the  solution 
and  is  subject  to  the  influence  of  temperature  as  stated  previously. 
Also  the  osmotic  pressure  of  a  given  quantity  of  substance  is  inversely 
proportional  to  the  volume  (p.  174).  When,  therefore,  a  solution  of  a 
great  concentration  is  separated  from  that  of  less  concentration  with  a 
semipermeable  membrane  between,  the  pressure  exerted  on  each  side  of 
the  membrane  will  be  proportional  to  the  concentration  of  the  solutions. 
The  pressure  will  be  influenced  by  temperature  and  there  will  be  a 
stirring  of  the  unequal  forces  to  gain  an  equilibrium.  If  only  the  solv- 
ent in  the  two  solutions  of  different  concentration,  as  just  referred  to, 
passes  the  membrane,  then  there  will  be  movement  toward  and  a  grad- 
ual dilution  of  the  more  concentrated  until  it  becomes  equalized  with 
the  other;  if  both  solvent  and  solute  pass  there  will  be  by  the  passage  of 
both  through  the  not  truly  semipermeable  membrane  an  effort  to 
equalize  with  more  or  less  exchange  from  both  solutions  as  in  the  case 
of  obstructed  diffusion. 

*  The  writers  call  especial  attention  to  Osterhout's  work  and  that  of  his  students  as  published 
in  the  Journal  of  General  Physiology,  Journal  of  Biological  Chemistry,  Science  and  the  Botanical 
Gazette. 


PHYSICAL  FORCES  INVOLVED  IN  BIOLOGICAL  ACTIVITIES      177 

In  the  discussions  of  osmotic  pressure  there  has  been  constantly  in 
mind  the  action  of  solutions  upon  microorganisms.  Either  a  cell-wall 
or  membrane  exists  as  a  distinct  structural  part  as  in  the  yeast  cell  or 
the  protoplasm  comes  in  contact  with  its  surrounding  medium  without 
any  distinctive  cell-wall  or  membrane  as  in  the  amoeba.  Whether 
there  is  a  layer  of  protoplasm  on  the  outer  surface  of  the  amoeba  which 
has  the  functioning  capacities  of  a  distinctive  cell- wall  may  not  be  easily 
asserted  for  there  is  evidence  pointing  to  the  two  possibilities.  Inas- 
much, however,  as  the  passage  of  materials  into  the  substance  of  the 
cell  is  really  that  of  diffusion  or  a  modification  of  it,  and  species  and 
varieties  respond  differently  to  this  diffusion,  it  is  easily  seen  that 
every  species  at  least  must  be  considered  by  itself  in  this  respect  and 
values  likewise  determined. 


FIG.  106. — Plasmolysis  in  cells  (After  DeVries  from  Macleod). 

It  is  well  known  that  water  will  pass  into  some  cells  and  cause  them 
to  swell  or  fill  out  when  apparently  the  substance  of  the  cell  or  its  fluid 
content  is  more  concentrated  than  the  surrounding  medium.  On  the 
other  hand,  when  the  medium  without  is  more  concentrated  than  the 
cell-contents,  water  flows  from  the  cell  toward  the  more  concentrated 
solution  outside  of  the  cell  and  accordingly  the  cell  shrinks.  This  is 
many  times  made  evident  by  the  contraction  of  the  protoplasm.  This 
process  in  which  the  water  is  abstracted  from  the  cell  through  osmotic 
pressure  is  known  as  plasmolysis. 

COLLOIDS  AND  CRYSTALLOIDS 

Since  the  time  of  Thomas  Graham  who  established  these  two 
classes  of  substances  there  has  been  a  growing  interest  in  them.  At 
present,  however,  instead  of  dividing  substances  into  two  classes 
placing  one  substance  in  one  cjass,  as  colloids,  and  another  distinct 

'      12 


178  PHYSIOLOGY   OF   MICROORGANISMS 

substance  in  the  second  class,  as  crystalloid,  one  and  the  same  substance 
may  exist  in  both  classes.  Therefore,  two  conditions  or  states  of  the 
same  substance  may  be  found,  the  one  the  colloidal,  the  other  the 
crystalloidal  condition  or  state.  Consequently,  substances  cannot  be 
divided  in  accordance  with  the  early  views  of  Thomas  Graham,  but 
the  conditions  or  state  under  which  they  exist,  may  be  so  divided  into 
colloids  and  crystalloids.  The  resolution  of  these  classes,  as  will  be 
seen,  is  fraught  with  many  difficulties. 

The  usual  ultimate  chemical  and  physical  conception  of  matter  is 
molecular  and  atomic.  Associated  with  this  are  physical  properties 
and  qualities.  Comparatively  recently,  matter  has  taken  on  new 
interpretations  for  the  molecule  and  atom  have  extended  to  the  electron 
and  sub-electron  possessing  definite  electric  potentialities.  In  the 
opposite  direction  there  appears  to  be  an  aggregating  or  massing  power 
along  with  the  solvent  belonging  to  the  molecule  in  which  the  atom  and 
electrons  may  be  active.  This  aggregating  power  does  not  seemingly 
manifest  itself  in  the  same  manner  with  all  substances;  in  other  words 
the  particle  resulting  from  this  aggregation  in  the  case  of  hydrated 
silicic  acid  may  not  be  executed  in  the  same  manner  as  in  the  case  of 
ferric  hydroxide;  in  the  case  of  gelatin,  as  in  the  case  of  casein;  in  the 
case  of  particulate  gold  as  in  the  case  of  particulate  carbon.  Such 
aggregate  particles,  apparently,  are  different  from  the  molecular  or 
atomic  particles  in  their  structures  and  reactions  and  the  term  aggre- 
gate does  not  convey  the  true  structural  nature.  In  molecular  reactions 
chemistry  follows  its  usual  course;  in  the  particulate  reactions,  physical 
manifestations  form  the  basis  of  recognition.  These  differentiations, 
while  helping  to  distinguish  between  the  well  known  structures  met  in 
crystalloidal  chemistry  and  the  more  or  less  amorphous  structures  of 
colloidal  chemistry  cannot  be  held  as  a  fast  cleavage  line  because  they 
merge  into  each  other  and  too  little  is  understood  of  the  structure  of 
colloids.  They,  however,  are  suggestive,  directive  and  helpful. 

Crystalloids  form,  as  a  rule,  true  molecular  or  ionic  solutions  (see 
Solutions,  p.  1 56)  while  colloids  form  solutions  of  a  more  or  less  mechani- 
cal character;  the  former  produce  a  uniformly  dispersed  homogeneous 
system  not  separable  mechanically,  the  latter  give  rise  to  a  solution 
mechanically  separable  and  not  uniformly  dispersed — a  heterogeneous 
system.  Also  the  former  give  rise  to  a  one-phase  system  while  the 
latter  yield  a  polyphasic  system.  The  solution  of  colloids  is  concretely 


a, 


PHYSICAL  FORCES  INVOLVED  IN  BIOLOGICAL  ACTIVITIES      179 


ustrated  by  reference  to  casein  in  milk,  or  gelatin  in  aqueous  solution, 
which  is  easily  grasped  to  differ  from  a  solution  of  salt,  a  crystalloid,  in 
water.  In  colloidal  solutions  the  particles  are  referred  to  as  the  disperse 
phase,  the  medium  in  which  they  are  found,  the  dispersion  means  and 
the  solution  as  a  whole,  a  dispersoid.  In  the  event  that  gold  be  reduced 
so  fine  that  its  suspension  gives  rise  to  a  colloidal  solution,  gold  would  be 
the  disperse  phase,  water  the  dispersion  means  and  the  solution  or 
suspension  as  a  whole,  the  dispersoid.  The  gold  would  also  represent 
one  phase  and  the  water  another  phase,  resulting  in  a  diphasic  hetero- 
geneous system.  Where  the  gold  particle  and  the  water  meet  or  at  the 
point  the  disperse  phase  and  dispersion  means  come  together  or  are  in 
contact  is  the  so-called  interface  so  important  in  surface  energy.  Some- 
times the  disperse  phase  is  called  the  internal  phase  and  the  dispersion 
means  the  external  or  continuous  phase. 

Dispersoids  exist  as  suspension-colloids  or  suspensoids  and  emulsion- 
colloids  or  emulsoids.  The  former  designate  the  disperse  phase  to  be  a 
solid  and  the  dispersion  means  a  liquid  (lyophobic  colloids)]  the  latter 
designate  the  disperse  phase  to  be  a  liquid  and  the  dispersion  means  also 
a  liquid  (lyophilic  colloids] .  As  an  example  of  the  former,  colloidal  gold 
as  the  disperse  phase  and  water  as  the  dispersion  means  is  satisfactorily 
typical;  as  an  example  of  the  latter,  gelatin  as  the  disperse  phase  and 
water  as  the  dispersion  means  qualifies,  although  the  gelatin  is  very 
close  to  a  solid  at  times  but  probably  still  in  a  hydrated  condition. 
This  attempt  to  divide  the  colloidal  condition  or  state  into  two 
classes  is  quite  general.  In  the  above  paragraph  Von  Weimarn*  and 
Ostwald  have  made  the  division  into  suspensoids  and  emulsoids, 
Perrinf  into  lyophobic  and  lyophilic.  NoyesJ  contributes  another 
division:  "As  types  of  these  I  would  draw  your  attention  to  these 
aqueous  solutions  of  gelatin  and  of  colloidal  arsenious  sulphide.  The 
former  class  possesses  a  much  greater  viscosity  than  that  of  water;  the 
latter  does  not  appreciably  differ  from  it  in  this  respect.  The  former 
gelatinizes  upon  cooling  or  upon  evaporation,  and  passes  again  into 
solution  upon  heating  or  addition  of  the  solvent;  the  latter  does  not 
gelatinize  upon  cooling,  and  if  gelatinized  by  other  means  it  does  not 
redissolve  upon  heating.  The  former  is  not  coagulated  by  the  addition 
of  salts  (unless  in  excessive  amount),  the  latter  immediately  gives  an 


*  Von  Weimarn,  Grundzuge  der  Dispersoid  Chemie  (Steinkopff,  Dresden),  1911. 

t  Perrin,  J.,  J.  Chim.  Phys.,  3,  SO,  1905. 

%  Noyes,  A.  A.,  Jour.  Amer.  Chem.  Soc.  27,  2,  p.  85,  1905. 


180  PHYSIOLOGY  OF  MICROORGANISMS 

abundant  precipitate.  We  have  therefore  to  distinguish  the  viscous, 
gelatinizing,  colloidal  mixtures,  not  coagulated  by  salts,  from  the  non- 
viscous,  non-gelatinizing,  but  readily  coagulable  mixtures.  The 
former  class  I  shall  designate  colloidal  solutions,  the  latter  colloidal 
suspensions"  Other  divisions  of  much  the  same  character  have  been 
suggested.  All  lack  in  fundamental  significance.  They  follow  much 
the  same  cleavage  line  but  it  possesses  a  ragged  fringe.  Whether  of 
great  or  permanent  value  or  not,  it  is  useful  until  a  more  definite,  basic- 
ally sound,  division  can  be  established. 

Colloidal  solutions  may  exist  in  which  the  disperse  phase  may  be 
found  in  other  dispersion  means  than  water.  These  with  water  are 
generally  known  as  sols.  When  the  dispersion  means  is  water,  the  so- 
lution or  suspension  is  specifically  called  hydrosol;  in  alcohol,  alcosol; 
in  glycerol,  glycersol;  etc.  If  the  disperse  phase  takes  up  a  certain 
amount  of  water,  it  may  enter  into  a  jelly-like  condition  when  it  is 
generally  called  a  gel.  In  this  instance,  it  would  be  called  specifically 
a  hydrogel.  It  is  possible  to  have  as  well  alcogels,  sulphogels,  etc. 
Gelatin  may  exist  in  a  colloidal  solution  as  a  hydrosol  and  also  as 
a  hydrogel  depending  upon  the  amount  of  water  employed.  There 
also  always  exists  the  possibility  of  the  disperse  phase  taking  up 
some  of  the  dispersion  means  and  the  dispersion  means  actually  in- 
corporating some  of  the  disperse  phase.  To  what  extent  this  may 
be  carried  is  problematical. 

It  has  already  been  indicated  that  colloidal  solutions  differ  from 
crystalloidal.  The  crystalloidal  solutions  are  true  molecular  or  ionic 
solutions.  The  molecule  may  or  may  not  divide  into  ions.  Sodium 
chloride  passing  into  solution  breaks  into  ions  carrying  with  them 
a  positive  and  negative  electric  charge  which  in  turn  create  a  cur- 
rent of  electricity.  The  cane  sugar  molecule  on  the  other  hand  does 
not  break  up  but  goes  into  a  molecular  solution;  there  are  no  positive 
and  negative  ions,  consequently  no  electric  dissociation.  Substances 
which  ionize  as  sodium  chloride  are  called  electrolytes  while  substances 
as  cane  sugar  are  non-electrolytes  because  they  do  not  ionize.  The 
colloids,  too,  like  sugar,  are  non-electrolytes  and  do  not  ionize,  yet  they 
respond  to  a  current  of  electricity  passed  through  a  solution.  The 
particles  of  a  colloid  have  a  tendency  to  pass  to  one  pole  or  the  other 
depending  upon  the  nature  of  the  colloid.  This  reaction  is  called 
electro phoresis.  Further,  it  may  be  said  that,  if  colloids  pass  toward 


PHYSICAL  FORCES  INVOLVED  IN  BIOLOGICAL  ACTIVITIES      l8l 


the  anode,  they  are  negatively  charged,  if  toward  the  cathode  positively 
charged.  The  significance  of  this  movement  of  the  particles  of  different 
colloids  in  response  to  an  electric  current  passed  through  a  solution  does 
not  seem  to  be  clearly  understood. 

The  size  of  the  particles  existing  in  a  suspensoid  or  an  emulsoid  or 
even  in  a  molecular  solution  is  of  considerable  importance  from  the 
standpoint  of  stability,  reaction  to  light  and  many  other  phenomena. 
Ostwald*  presents  the  matter  very  tersely  in  the  following  diagram 
which  has  been  slightly  modified  by  the  writers. 


Dispersoids 


True  or  coarse 

dispersions 

(suspensions,  emulsions, 
etc.) 

Size  of  the  particles  of 

disperse  phase  greater 

than  O.IM 


Colloidal  solutions 

(Suspensoids,  emulsoids, 

etc.) 

Size  of  particles  of  the 

disperse  phase  between 

o.ifj,  and  i  fin 


\ 

Molecular  or 
supermolecular 
dispersoids 

Size  of  particles  of  the 

disperse  phase  about 

i  nn  or  less 


Colloidality  decreases 


Degree  of  dispersion  increases. 
FIG.  107. — An  arrangement  of  dispersoids.     (After  Ostwald.) 

This  graphic  presentation  can  be  still  better  understood  by  giving  also 
the  illustration  provided  on  page  30  of  the  same  publication  (Fig.  8) 
of  this  publication  (Fig.  108). 

By  use  of  the  ultramicroscope  developed  by  Siedentopf  and  Zsig- 
mondy  it  has  been  possible  to  employ  Tyndall's  phenomenon  which 
makes  the  visibility  of  rays  of  light  passing  through  a  medium  depend- 
ent upon  solid  particles  as  dust  in  the  air  of  a  room.  The  light  must 
enter  into  a  dark  room  as  a  ray  from  one  side  only  to  illuminate  the 
particles  and  render  the  demonstration  successful.  In  the  same  man- 
ner particles  suspended  in  a  transparent  medium  may  also  be  illumin- 

*  Ostwald,  Wolfgang.    Handbook  of  Colloid  Chemistry,  p.  33. 


182 


PHYSIOLOGY   OF   MICROORGANISMS 


ated.  The  ultramicroscope  makes  it  feasible  to  use  Tyndall's  phe- 
nomenon effectively  in  revealing  particles  of  some  colloidal  substances 
and  solutions  having  particles  of  larger  dimensions.  Siedentopf  and 


Anthrax 
bacillus 

about 
6u  long 


Particles  oF  colloid  gold 


D  Precipitated  parHde 
of  gold,  about-  75fJ)j 


Starch     Chloroform  Hydrogen 
\molecule     molecule    molecule 


\Enlamemenr.lOOOOOO  tot 


Parfides  of  a  fine  mastic 


Enlargement-  3333  fo  1 


FIG.  108. — Comparison  of  particles  of  different  sizes.     (Ostwdd.} 

The  large  circle  corresponds  to  the  diameter  of  a  human  red  blood  corpuscle 
(about  7.5  M);  the  large  pentagon  to  that  of  a  starch  granule  of  medium  size  (about 
7.0  /t).  The  particles  enclosed  in  a  frame  are,  in  comparison  with  the  rest  of  the 
figure,  enlarged  333  times. 

The  figure  has  been  constructed  from  data  and  tables  given  in  R.  Zsigmondy 
(Zur  Erkenntnis  der  Kolloide,  Jena,  1905).  The  values  for  the  mastic  suspension 
are  taken  from  /.  Pcrtin's  studies  [Kolloidchem.  Beihefte  I,  221  (1910)]. 


Zsigmondy  find  that  the  microscope  has  its  limitation  of  visibility  at 
about  o.i/x  and  the  ultramicroscope  at  about  I.G//JU  (sulmicrori)  or 
o.ooi;u.  There  are  particles  existing  beyond  the  reach  of  the  ultra- 


PHYSICAL  FORCES  INVOLVED  IN  BIOLOGICAL  ACTIVITIES      183 

microscope  which  are  designated  in  size  by  the  term  amicrons.     Accord- 
ing to  Zsigmondy  the  size  of  the  particles  covered  in  colloids  ranges 


FIG.  ioga. — Arrangement  of  ultramicroscope.     (After  Bayliss.) 


FIG.  1096. — Rays  of  light  in  ultramicroscope.     (After  Bayliss.) 


from  o.i /x  to 
cules : 


Ostwald  gives  the  estimated  sizes  of  certain  mole- 


Hydrogen  gas 0.067-0. 1 57/iM 

Water  vapor o .  i  I^H/JL 

Carbon  dioxide o .  285/x/x 

Sodium  chloride o .  26/x/x 

Sugar Q.7MM 

Some  cpnception  of  the  size  of  molecules  and  colloidal  particles, 
although  they  may  not  be  absolute  and  even  subject  to  great  range  or 
variability,  contributes  to  an  understanding  of  colloidal  and  molecular 
solutions,  osmotic  action,  life-activities,  lower  limits  of  size  of  micro- 
organisms and  other  natural  phenomena. 

The  "disperse  phase"  of  colloidal  solutions  suggests  at  once  the 
extensive  surface  made  possible  by  the  particles  in  suspension  and  must 
likewise  suggest  the  extent  of  surface  energy  present  in  the  form  of 
surface  tension  and  adsorption.  These  factors  are  largely  involved  in 


184  PHYSIOLOGY   OF   MICROORGANISMS 

colloidal  reactions  and  life-functions.  Their  bearing  has  been  already 
indicated  (See  168). 

It  has  already  been  said  that  Thomas  Graham  made  the  distinction 
between  colloids  and  crystalloids  by  means  of  dialysis  through  a  mem- 
brane, the  colloids  are  withheld  and  crystalloids  pass  through.  This 
movement  on  the  part  of  these  substances  follows  the  laws  of  diffusion 
which,  in  turn,  conform  with  the  laws  of  expansion  of  gases.  In  the 
case  where  the  membrane  obstructs  the  movement  of  colloids  and 
permits  the  crystalloids  to  pass  there  can  be  recognized  an  interference 
with  free  movement.  Whether  the  colloidal  molecule  is  larger  than 
the  crystalloidal  molecule,  which  appears  to  be  a  fairly  satisfactory 
undemonstrated  reason,  or  not,  does  not  materially  alter  the  situation; 
or  whether  some  chemical  transition  or  obstruction  accounts  for  this 
phenomenon  of  passage  and  check,  in  our  present  position,  does  not 
contribute  much  without  a  real  working  knowledge  of  what  is  involved. 
The  facts  remain:  Colloidal  substances  do  not  pass  while  crystalloids 
do.  This  significant  condition  may  be  actually  responsible  for  the 
cell-entities  which  incorporate  the  mechanism  of  life. 

In  colloids,  diffusion  is  slow,  slower  than  in  the  case  of  the  crystal- 
loids. This  enables  the  crystalloids  to  penetrate  or  diffuse  through  the 
colloidal  substances  as  protoplasm  and  sustain  what  must  be  regarded 
as  a  more  or  less  fixed  substance,  protoplasm,  through  the  very  nature 
of  its  powers. 

The  microbial  cell  is  generally  a  unicellular  organism  which  secures 
its  nutrition  and  performs  its  respiratory  functions  through  the  surface 
layer  of  the  cell.  This  outer  layer  in  most  microbial  cells  takes  the 
form  of  a  membrane  and  where  no  membrane  exists  the  cell  seems  to 
respond  in  much  the  same  manner  through  its  protecting  surface  layers 
of  protoplasm.  A  yeast  cell  prepares  its  food  which  is  not  assimilable 
through  its  cell- wall  by  secreting  suitable  enzymes  to  produce  diffusible 
nutrition.  Such  portions  of  this  solution  are  assimilated  through  the 
cell-wall  as  are  needed  in  cell-construction  and  are  converted  by  similar 
processes  within  the  cell  substance  while  in  transitional  route  to 
protoplasm  itself.  In  the  case  of  an  amceba  the  particle  of  food  is 
often  taken  within  the  protoplasm  by  means  of  its  pseudopodia  and 
after  digestion  is  assimilated  as  in  the  yeast  cell.  This  process  in  the 
amoeba  cannot  be  regarded  as  at  all  different  from  that  of  the  yeast  for 
the  digestive-preparatory  process  and  assimilation  are  much  the  same. 


PHYSICAL  FORCES  INVOLVED  IN  BIOLOGICAL  ACTIVITIES      185 

When  food  is  prepared  it  is  probably  in  the  form  of  a  molecular  or 
ionic  dispersoid  which  enters  the  substance  of  the  protoplasm  and 
diffuses  readily.  The  ionization  of  the  cell  is,  according  to  many 
authorities,  dependent  upon  the  ionic  or  molecular  dispersoids  which 
are  found  in  the  cell  substance,  whether  they  are  on  their  way  to  become 
protoplasm  or  are  the  products  of  cell  activity.  When  these  ionic  or 
molecular  dispersoids  of  the  cell  are  of  a  nature  and  possess  the  affinity 
to  attach  themselves  to  molecules  of  protoplasmic  structure,  their 
diffusibility  is  lost  and  they  become  anchored;  if,  however,  there  exist 
diffusible  substances  which  are  cast  off  from  the  protoplasmic  molecules 
by  metabolic  action  and  no  longer  possess  the  affinity  for  attaching 
themselves,  their  dissipation  by  elimination  is  -assured.  The  change  of 
starch,  glycogen,  protein,  as  food,  to  diffusible  products  by  regulation 
digestive  processes  and  the  elimination,  as  waste  products,  of  diffusible 
substances  have  a  tendency  to  confirm  this  vital  interpretation. 

Literature  freely  consulted  and  recommended  for  extended  study. 

BAYLISS,  The  Principles  of  General  Physiology. 

BURTON,  Physical  Properties  of  Colloidal  Solutions. 

CLARK,  W.  M.,  The  Determination  of  Hydrogen  Ions,  1920. 

HATSCHEK,  Colloids. 

HOBER,  Physikalische  Chemie  der  Zelle  und  der  Gewebe. 

ITANO,  The  Relation  of  Hydrogen  Ion  Concentration  of  Media  to  the  Proteolytic 

Activity  of  B.  subtilis. 
JONES,  Nature  of  Solutions. 
KIMBALL,  College  Physics. 

MACLEOD,  Physiology  and  Biochemistry  in  Modern  Medicine. 
McCLENDON,  Physical  Chemistry  of  Vital  Phenomena. 
MICHAELIS,  L.,  Die  Wasserstoffionenkonzentration. 
NICHOLS  and  FRANKLIN,  The  Elements  of  Physics. 
NORTHRUP,  Laws  of  Physical  Science. 
OSTWALD-FISCHER,  Handbook  of  Colloidal  Chemistry. 
PERRIN,  Brownian  Movement  and  Molecular  Reality. 
PHILIP,  Physical  Chemistry. 

SORENSEN,  S.  P.  L.,  Ergebnisse  d.  Physiologie,  Bd.  12,  1912. 
VON  PROWAZEK,  Physiologie  der  Einzelligen. 
THOMSON,  The  Corpuscular  Theory  of  Matter. 
THOMSON,  Rays  of  Positive  Electricity. 
WALKER,  Introduction  to  Physical  Chemistry. 
WASHBURN,  Principles  of  Physical  Chemistry. 
WELLS,  Chemical  Pathology. 


CHAPTER  III 

CHEMICAL   STUDIES  OF   THE   CONTENT   OF   MICROBIAL 

CELLS* 

Microorganisms  have  a  widely  variable  chemical  composition. 
They  differ  so  much  in  their  requirements — their  habits,  their  food 
needs,  their  moisture  demands,  their  environmental  atmosphere,  and 
their  capacity  for  change — that  their  great  deviation  from  a  constant 
nature,  as  manifested  by  superficial  expressions,  perhaps,  does  not 
awaken  unexpected  mental  responses.  They  also  undergo  much 
alteration  in  their  compositional  nature  as  well  as  in  their  structural 
nature  while  passing  stages  in  their  individual  developments.  The 
vegetative  or  growing  forms  do  not  seem  to  have  the  same  composition 
as  the  spore-forms  or  resting  forms  although  it  may  be  quite  possible 
that  fundamentally  the  exact  composition  exists  in  both  and  only  more 
superficial  substances  are  detectable;  old  cells  differ  from  young  cells 
and  capsulated  forms  from  uncapsulated  forms.  Food  influences 
greatly  the  products  found  in  protoplasm  both  quantitatively  and 
qualitatively.  While  such  products  which  are  referable  to  food  may 
not  be  strictly  a  part  of  what  is  contemplated  in  the  composition  of  the 
cell,  yet  it  is  difficult  many  times  to  make  the  distinction.  Doubtless 
most  influencing  agents  whether  external  or  internal  have  some  power 
over  the  substances  now  recognized  in  cellular  composition. 

If,  however,  constancy  in  species  is  to  be  maintained,  it  is  necessary 
to  assume  that  there  is  to  be  found  in  every  species  a  constant  group  or 
nucleus  of  chemical  atoms  or  molecules  whether  existing  independently 
or  acting  in  consort  in  forming  congeries  of  molecular  complexes,  and 
that  substances  fluctuating  in  their  presence  or  in  their  amount  must 
be  regarded  as  more  incidental  to  the  basic  life-processes.  Species, 
therefore,  even  when  undergoing  all  the  recognized  variations  to  which 
it  is  subjected — ageing,  developmental  stages,  reproduction,  environ- 
mental factors  as  food,  reaction,  oxygen  supply,  temperature,  and  others 

*  Prepared  by  Charles  E.  Marshall  and  Arao  Itano.. 

1 86 


CHEMICAL  STUDIES  OF  THE  CONTENT  OF  MICROBIAL  CELLS    187 

—remains  basically  constant,  apart  from  its  evolutionary  possibilities, 
to  its  line  of  descent. 

The  student,  too,  should  not  be  led  to  interpret  the  products  found 
by  the  chemists  as  the  substances  constituting  protoplasm  or  any  of  its 
differentiated  parts  but  rather  as  substances  entering  into  the  formation 
of  the  protoplasmic  molecule,  or  as  substances  resulting  from  metabolic 
processes,  or  as  substances  connected  in  some  way  with  the  food  supply 
as  reserve  material  or  as  substances  essentially  foreign,  having  entered 
the  cell  by  means  of  its  mechanical  functional  acts.  Ultimate  analyses 
may  reveal  the  percentages  of  N,  C,  H,  O,  P,  S  and  other  elements; 
certain  chemical  methods  may  demonstrate  the  presence  of  proteins, 
amino  acids,  carbohydrates,  and  fats,  and  the  ash  may  contain  definite 
mineral  constituents,  yet  such  revelations  are  only  the  initial  steps 
which  will  take  the  wandering  industrious  scientist  or  student  to  the 
museum  of  nature  wherein  are  found  the  depicted  substances  and  acts 
involved  in  living  protoplasm.  However,  besides  striving  to  obtain 
an  insight  into  the  very  nature  of  life  and  its  operating  processes,  much 
has  been  accomplished  by  such  studies  in  ameliorating  the  conditions  of 
man's  existence  and  in  helpfulness.  By  having  even  this  very  limited 
knowledge  as  will  be  gathered  from  the  study  of  metabolism,  soil,  food, 
immunity  and  infectious  diseases,  extending  to  agriculture,  medicine 
and  the  industries,  great  progress  is  possible  and  has  been  made. 

ANALYSES 

Moisture. — The  moisture  content  of  microorganisms  has  a  very 
wide  range.  In  the  mother-of-vinegar  made  up  largely  of  acetic 
bacteria,  the  moisture  content  reaches  98.3  per  cent.;  in  Bad.  pneu- 
monia,* 85.55  per  cent.;  in  the  alga,  Chlorella  vulgaris,*  63.06  per  cent.; 
in  the  spores  of  molds,  39  to  44  per  cent.  From  this  very  brief  survey 
it  will  be  seen  that  all  microorganisms  vary  greatly  in  their  moisture 
content.  The  amount  seems  to  be  largely  dependent  upon  the  medium 
in  which  development  takes  place,  unless  it  is  in  the  case  of  spores  which 

*  Nicolle,  M.,  and  Alilaire,  E.,  in  Ann.  Inst.  Pasteur,  T.  23,  p.  555,  furnishes  the  following 
moisture  determinations  in  per  cent.:  Bad.  mallei,  76.49;  Bad.  cholera  gallinarum,  79.35; 
Msp.  comma  (Bombay),  73.38;  Bad.  dysenteries  (Shiga),  78.23;  Proteus  vulgaris  (B.  proteus), 
79.99;  B.  typhosus,  78.93;  Bad.  anthracis  (asporogenic) ,  81.74;  Bad.  pseudotuberculosis,  78.83; 
Bad.  pneumonia,  85.55;  B.  coli,  73.35;  B.  prodigiosus,  pathogenic  (de  Fortineau),  78.00;  B. 
psittacosis,  78.05;  Bad.  diphtheria,  84.50;  B.  pyocyaneus,  74-99;  B.  lymphangitis  (de  Nocard), 
77.90;  yeast  (Frohberg),  69.25;  Chlorella  vulgaris  (alga),  63.6. 


1 88  PHYSIOLOGY   OF   MICROORGANISMS 

incorporate  an  amount  which  is  difficult  to  remove  and  which  has 
some  relation  apparently  to  their  high  degree  of  resistance. 

Molds  have,  as  a  rule,  a  greater  moisture  content  than  yeast  and 
yeast  a  greater  content  than  bacteria,  yet  these  organisms  have  no 
constancy  or  uniformity  in  their  moisture  content.  The  protozoal 
forms  are  as  dissimilar  as  others  and  their  range  of  moisture  content 
assumes  no  fixed  boundaries. 

Although  there  is  a  minimum  limit  and  a  maximum  limit  as  indi- 
cated on  the  one  hand  by  desiccation  and  on  the  other  hand  by  an 
inability  to  absorb  more  moisture,  still  retaining  life  one  is  forced  to 
believe  in  a  very  restricted  amount  of  moisture  as  essential  to  life- 
processes.  Beyond  this  essential  amount,  in  the  case  of  too  little,  the 
metabolic  activities  cannot  take  place,  and,  in  the  case  of  an  excessive 
amount,  proper  functioning  is  interfered  with  or  a  modification  of 
physiological  reactions  gradually  becomes  more  and  more  evident. 

Proteins  and  other  nitrogenous  substances. — Nitrogenous  compounds 
are  present  in  varying  amounts  and  are  assumed  to  be  the  basis  of 
protoplasm.  The  approach  in  the  study  of  this  class  of  substances 
has  been  made  through  the  determination  of  nitrogen,  then  converting 
the  nitrogen  into  terms  of  protein  by  the  use  of  the  recognized  factor; 
by  the  recognition  of  definite  nitrogenous  compounds  which  may 
represent  certain  portions  of  the  protein  molecule;  and  by  the  use  of 
reagents  long  employed  to  detect  the  presence  of  protein,  largely 
qualitatively.  All  of  these  can  furnish  only  inadequate  means  for  the 
recognition  of  the  nitrogenous  materials  which  may  enter  into  the 
formation  of  the  active  life-substance,  protoplasm.  However  limited 
may  be  the  knowledge  available  in  this  particular  subject,  there  is  now 
at  hand  sufficient  to  point  the  way  for  more  and  for  certain  directive 
practical  purposes.  The  per  cent,  of  nitrogen  *  found  by  Vaughan  and 
his  associates  and  by  Nicolle  and  Alilaire  ranges  from  3.96  (dry  weight, 

*  Vaughan  and  Wheeler.  "Protein  Split  Products  in  Relation  to  Immunity  and  Disease," 
by  Vaughan,  contributes  the  nitrogen  determinations  in  per  cent,  for  several  bacteria:  Typhoid, 
11.55;  colon,  10.65;  tuberculosis,  10.55 ;  anthrax,  10.285;  subtilis,  5-964;  Proteus  vulgaris,  6.791; 
Ruber  of  Kiel,  10.655 ;  megaterium,  8.349;  pyocyanus,  10.843;  violaceus,  11.765;  Sarcina 
aurantiaca,  11.46. 

Nicolle,  M.,  and  Alilaire,  E.,  in  Ann.  liist.  Pasteur,  23,  555,  give  the  following  nitrogen  re- 
sults in  per  cent,  (based  upon  dry  weight),  Bact.  mallei,  10.47;  Bad.  cholera  gallinarum,  10.79; 
Msp.  comma  (Bombay),  9.79;  Bact.  dysenteries  (Shiga),  8.89;  B.  proteus  (Proteus  vulgaris), 
10.73;  B.  typhosus,  8.28;  Bact.  anthracis  (asporogenic) ,  9.22;  Bact.  pseudotuberculosis,  10.36; 
Bact.  pneumonias,  8.33;  B.  coli,  10.32;  B.  prodigiosus  (pathogenic)  (de  Fortineau),  10.55;  B. 
psittacosis,  9.55;  B.  pyocyaneus,  9.79;  B.  lymphangitis  (de  Nocard),  9.17;  yeast  (Frohberg), 
10. oo;  Chlorella  vulgaris,  3.96. 


CHEMICAL  STUDIES  OF  THE  CONTENT  OF  MICROBIAL  CELLS    189 


in  Chlorella  vulgaris  (alga)  to  10.73  m  B-  proteus  (Proteus  vulgaris). 
In  the  protozoon,  Noctiluca  miharis,  there  was  present  7. 74  per  cent,  of 
nitrogen  as  determined  by  Emmerling.*  Molds  and  yeasts  appear  to 
lie  between  the  alga  named  and  many  of  the  bacteria  as  indicated  by  the 
work  of  Marschall  and  Nageli.f 

The  compounds  of  nitrogen  which  have  been  determined  are  quite 
numerous  although  it  must  be  allowed  that  the  analyses  have  not  always 
been  satisfactory.  Ruppelf  claims  to  have  determined  nucleic  acid, 
nucleoprotamin,  nucleoproteid,  albuminoids  (keratin,  etc.)  in  dried 
Bact.  tuberculosis.  Nishimura||  found  nuclein  bodies  as  xanthin, 
guanin,  adenin  in  a  water  bacillus.  Vaughan§  and  his  associates  have 
been  able  to  demonstrate  the  presence  of  various  amino  acids.  The 
work  of  Emmerling*  also  contributes  much  which  aids  in  our  under- 
standing of  definite  substances  in  the  protoplasm  of  protozoa. 

*  Emmerling,  O.,  Biochem.  Zeitschr.,  1909,  gives  this  analysis  of  Noctiluca  miharis:  In  100 
grams  of  ash  free  substance  there  was  7-74  grams  of  nitrogen  (Taken  from  S.  von  Prowazek: 
Physiologie  der  Einzelligen.) 

Lysin o .  212    with  o  .040  grams  nitrogen 

Arginin 1 .6492  with  o  .432  grams  nitrogen 

Histidin 3  .4762  with  0.938  grams  nitrogen 

Tyrosin 0.5271  with  0.041  grams  nitrogen 

Glycocoll 15  .9000  with  2  .956  grams  nitrogen 

Alanin 2  .4000  with  o  .378  grams  nitrogen 

Leucin o  .4200  with  o  .044  grams  nitrogen 

Prolin 4 . 6000  with  o .  556  grams  nitrogen 

Asparagin  acid, o .  1700  with  o  .020  grams  nitrogen 


Total S  -  405  grams  nitrogen. 

t  Marschall,  Arch.  f.  Hyg.,  28,  19,  estimates  the  protein  in  Aspergillus  at  30.4  per  cent.,  in 
Penicillin.™,  at  40.2  per  cent.,  and  Mucor  at  43.4  per  cent,  (based  upon  dry  weight).  In  Arch. 
f.  Hyg.  28,  1917,  17,  the  per  cent,  of  protein  in  molds  is  placed  at  38.0. 

Nageli  and  Loew.,  Jour.  Prakt.  Chem.  N.  P.,  17,  determined  47.0  per  cent,  of  protein  in 
yeasts. 

t  Ruppel.  Zeit.  f.  Physiol.  Chemie,  XXVI,  1898,  out  of  100  grams  of  dried  Bact.  tuberculosis 
secured  the  following  subs-tances: 

Nucleic  acid  (tuberculinic  acid) 8.5  grams 

Nucleoprotamin 25.5  grams. 

Nucleoproteid 23.0  grams 

Albuminoids  (keratin,  etc.) 8.3  grams 

Fatty  matter 26.5  grams 

Ash 9.2  grams 

.    !|  Nishimura,  Arch.  f.  Hyg.  XVIII,  318,  1893,  reports  the  finding  of  0.17  per  cent,  xanthin, 
0.08  per  cent,  adenin  and  0.14  per  cent,  of  guanin  in  his  water  bacillus. 

§Vaughan,  V.  C.  and  associates,  loc.  cit.,  have  noted  the  presence  of  certain  diamino  and 
monamino  acids. 


I QO  PHYSIOLOGY   OF   MICROORGANISMS 

The  protein  substances  vary  in  amount  in  different  species  of  micro- 
organisms. Vaughan*  compares  the  compounds  of  B.  coli  and  Bad. 
tuberculosis  indicating  that  no  similarity  of  amino  acids  exists  in  the 
protoplasm.  Duclauxf  has  found  in  the  analysis  of  yeast,  15  years  old, 
only  2.7  per  cent,  of  nitrogen  as  compared  with  the  yeast  (Frohberg) 
analyzed  by  Nicolle  and  Alilaire  which  contained  10  per  cent,  nitrogen. 
Age,  it  seems  from  this,  changed  the  amounts  of  nitrogenous  material 
present  in  the  cell.  Then,  again,  the  medium  upon  which  the  micro- 
organisms are  cultivated  has  a  decided  influence.  Cramer  J  determined 
69.25  per  cent,  protein  in  Msp.  comma  when  grown  in  bouillon  and  only 
35.75  per  cent,  when  grown  in  Uschinsky's  solution.  He  also  noted 
that  the  dry  matter  from  this  organism  was  greater  when  grown  at 
body- temperature  than  when  grown  at  room- temperature. 

Carbohydrates. — Substances  which  correspond  to  the  reactions  of 
carbohydrates  have  been  recognized.  Some  of  these  substances 
exist  as  distinctive  carbohydrates  and  some  enter  into  the  formation 
of  compounds  as  gly co-proteins.  Their  relation  to  the  protoplasmic 
molecular  structure  and  to  nutritive  processes  is  still  more  obscure. 

Glycogen  has  been  reported  by  A.  Fischer ||  in  B.  subtilis  and 
B.  coli.  Levene§  has  found  it  in  Bact.  tuberculosis.  Marschall  in  the 
study  of  molds  records  the  presence  of  3.7  per  cent,  starch.  How- 
ever, glycogen  is  so  much  like  starch  that  confusion  has  arisen. 
Glycogen  in  molds  and  yeasts,  much  like  that  of  animal  glycogen, 
is  cla'med  by  several  workers.  (Glycogen  has  been  commonly 
known  as  animal  starch  from  the  time  of  Claude  Bernard.)  In  proto- 
zoa glycogen  has  been  determined  by  SosnowskiU  mParamecium  and 
by  Biitschli  in  Gregarina. 

*  Vaughan,  V.  C.  and  his  associates,  loc.  cit.,  compare  the  amino  acids  of  B.  coli  and  Bact. 
tuberculosis. 

B.  coli,  Bact.  tuberculosis, 

Per  cent.  Per  cent. 

Glutanic  acid 3  -  oo  0.20 

Glycocoll 0.33  o .  oo 

Alanin '. i .  oo  i .  40 

Valin .' i.  60  4.60 

Leucin 2.00  1.82 

Phenylalanin : 0.20  0.50 

fDuclaux,  E.:  Kruse,  "Allgemeine  Mikrobiologie,"  p.  59. 

tCramer,  E.,  Arch.  f.  Hyg.  28,  i. 

||Fischer,  A.:  Vorlesungen  iiber  die  Bakterien,  Jena,  1903. 

§Levene,  Jour.  Med.  Research,  6,  135,  1901.     Scheibler,  Zeitsch.  f.  Rubenzuckerindustriei 
XXIV,  309,  1874,  Marschall,  Arch.  f.  Hyg.,  28,  19,  189?- 

HSosnowski,  Centralblatt  f.  Physiologic,  13,  1899. 


CHEMICAL  STUDIES  OF  THE  CONTENT  OF  MICROBIAL  CELLS    IQI 

Cellulose,  so  bound  up  with  plant  life  and  at  one  time  so 
much  used  to  differentiate  plant  and  animal  life,  has  not  been 
positively  demonstrated  in  any  microorganism,  even  in  molds  and 
yeasts.  Substances,  giving  suggestive  reactions,  have  been  studied 
and,  at  times,  have  been  called  cellulose,  or  some  modified  form  of 
cellulose,  yet  recent  analysts  seem  to  think  there  is  really  no  substantial 
ground  for  this  assumption.  Vaughan*  in  his  extensive  analyses  of 
bacterial  cells  has  never  been  able  to  identify  cellulose.  On  the  other 
hand  Vaughan  calls  attention  to  two  carbohydrate  bodies,  one  of  which 
furnishes  a  reducing  sugar  when  boiled  with  dilute  mineral  acid  and 
the  other  does  not. 

From  time  to  time  there  have  been  detected  suggestive  traces  of 
various  carbohydrate  substances  to  which  special  names  have  been 
attached  but  they  seem  to  lack  definiteness  and  individuality  in  their 
chemical  features.  Chitin,f  a  substance  quite  generally  found  in 
microbial  cell-walls,  consists  apparently  of  a  carbohydrate-amine  or 
glucosamine  polymerized.  Much  emphasis  is  now  placed  upon  this 
substance  as  representing  the  most  important  constituent  not  only  of 
microbial  cell-walls  but  of  wings  and  coverings  of  insects  and  of  many 
lower  animal  forms. 

Fats. — Many  analyses  indicate  variable  amounts  of  fat  in  all  classes 
of  microorganisms.  Whether  this  fat  is  the  result  of  degradation  proc- 
esses at  times,  whether  it  may  be  ready  for  assimilation,  whether  it 
exists  as  a  reserve  product,  or  whether  it  is  the  yield  of  direct  absorption 
cannot  be  asserted  off-hand.  Probably  there  are  times  when  it  may 
answer  to  each  of  these  explanations  and  times  when  indications  are 
such  as  to  furnish  a  positive  understanding. 

Fat  globules  may  be  readily  revealed  by  the  use  of  certain  stains 
as  osmic  acid  and  Sudan  III  when  present  in  comparatively  large 
microbial  cells,  but  in  the  case  of  bacterial  cells  this  procedure  is  un- 
availing, making  it  necessary  to  employ  recognized  chemical  methods. 

In  the  analysis  of  molds,  MarschallJ  has  obtained  the  following 

Aspergillus       Penicillium  Mucor 

Ether  extract 4.7  4.1  4.0 

Alcoholic  extract 18.5  n.8  n.8 

'Vaughan,  V.  C.  and  his  associates,  loc.  cit. 

tChitin  when  hydrolyzed  yields  glucosamine  and  acetic  acid.     The  equation  CisHsoNzOiz  + 
4H;0  =  2CH2OH.CHOH.CHOH.CHOH.CHNH2.CHO  +  3CHaCOOH,  has  been  suggested. 
JMarschall,  Arch.  f.  Hyg.,  28,  19,  1897. 


1 92  PHYSIOLOGY   OF   MICROORGANISMS 

results  from  the  ether  and  alcoholic  extracts  in  terms  of  per  cent,  of 
dry  substance.  Nageli  and  Loew*  found  5  per  cent,  in  a  bottom- 
fermentation  beer  yeast.  The  Bact.  tuberculosis  has  always  occupied 
a  conspicuous  place  on  account  of  its  fat-content.  Klebsf  estimated 
20.5  per  cent,  of  a  red  fat  and  1.14  per  cent,  of  a  white  fat.  In  amosbae, 
fat  globules  are  frequently  detectable  in  very  large  numbers. 

Apparently  the  fatty  materials  found  in  different  organisms  are  of 
diverse  natures.  Hammerschlag  J  believed  most  of  the  fatty  substances 
of  Bact.  tuberculosis  consist  of  tripalmitin  and  tristearin.  De  Schweinitz 
and  Dorset  ||  obtained  palmitic  and  arachidic  acids.  Bandraus§  recog- 
nizes stearin  and  olein  together  with  the  lipoids,  cholesterin  and  lecithin, 
in  the  same  species.  It  is  a  matter  of  determination  that  stearin, 
palmitin,  cholesterin,  lecithin  have  also  been  recognized  in  molds, 
yeasts,  and  protozoa.  There  is  no  characteristic  uniformity  existing 
between  species  other  than  certain  fatty  substances  are  more  commonly 
met  with  in  some  than  others.  In  the  same  species  the  fat  content  or 
amount  is  subject  to  wide  variations.  It  was  noticed  by  Meyer  ^f  that 
in  B.  tumescens  there  was  an  increase  of  fat  till  spore  production  when 
the  fat  completely  disappeared.  There  was  no  fat  in  the  spores. 

The  Ash  Elements. — It  is  exceedingly  difficult  at  the  present  time  to 
determine  the  number,  kinds  and  limitations  of  inorganic  elements 
included  in  the  compositional  structure  of  protoplasm.  '  Both  qualita- 
tive and  quantitative  studies  fail  in  solving  the  values  and  relationships 
of  these  elements  in  vital  processes.  From  the  nutritional  viewpoint  t 
certain  elements  may  be  recognized  as  very  important  and  others  as 
incidental.  Uniformity,  however,  exists  only  within  certain  bounda- 
ries, if  it  exists  at  all.  The  elements  which  stand  out  most  con- 
spicuously are  phosphorus,  potassium,  sodium,  calcium,  sulphur, 
magnesium,  iron,  silicon,  but  manganese,  aluminum,  copper  and  others 
have  been  recognized  at  times. 

The  finding  of  an  element  does  not  establish  its  relation  to  proto- 
plasmic synthesis.  Attempts  have  been  made  to  substitute  other 
elements  for  those  considered  essential  but  such  efforts  cannot  be 

*Nageli  and  Loew,  Sitzgsber.  d.  Kgl.  Academie  d.  wiss.  in  Munchen,  1878. 
fKlebs,  Cent.  f.  Bakteriologie,  XX,  488,  1896. 

JHammerschlag,  Monats  f.  Chem.,  X,  9,  1899;  Cent.  f.  Klin.  Med.,  XII,  9,  1891. 
||De  Schweinitz  and  Dorset,  Jour.  Amer.  Chem.  Soc.,  XVII,  605,  1895;  XVIII,  449,  1896 
XIX,  782,  1897;  XX,  618,  ia98. 

§Bandraus,  Compt.  rend.  ac.  sc.,  142,  657,  1906. 
IfMeyer:  Flora,  432,  1889. 


CHEMICAL  STUDIES  OF  THE  CONTENT  OF  MICROBIAL  CELLS    IQ3 


regarded  on  the  whole  as  eminently  satisfactory.  Illustrating,  no 
comment  is  needed  to  place  nitrogen  in  its  many  connections  and 
phosphorus  seems  to  be  very  intimately  bound  up  with  the  complex 
molecule  of  protein,  yet  when  potassium  and  iron  are  considered  it  may 
be  far  more  difficult  to  formulate  definite  conceptions  of  relationships. 
It  is  safe  to  say,  however,  that  ash  constituents  are  required  in  life- 
processes  even  if  a  more  detailed  analysis  is  barred  or  blurred  for  the 
time  being. 

The  extent  to  which  ash  elements  are  found  is  well  set  forth  by 
Kruse  *  in  a  comprehensive  review  in  which  he  considers  molds,  yeasts 
and  bacteria.  In  the  analyses  presented,  phosphoric  acid  appears  to 
exist  in  greater  proportion  than  all  other  elements.  Potassium  and 

*Kruse's  review  is  here  offered  in  abbreviated  form  (Allgemeine  Mikrobiologie,  pp.   86—87). 
Zopf  (Pilze,  1  1  8).     Higher  Molds. 

Phosphoric  acid  .........................................  40  .  o  per  cent. 

Potassium  .............................................  45  .  o  per'cent. 

Sodium  ................................................  1.4  per  cent. 

Magnesium  ............................................  2.0  per  cent. 

Calcium  .................................  .  .............  1.5  per  cent. 

Silicic  acid  .............................................  i  .  o  per  cent. 

Iron  oxide  .............................................  i  .  o  per  cent. 

Sulphuric  acid  ..........................................  8.0  per  cent. 

Chlorine  ...............................................  i  .  o  per  cent. 

Mayer,  Ad.  Garungschemie,  Aufl.,  5,  118,  1902.     Yeast. 

Phosphoric  acid  ......................................  51.0  -59  .  o  per  cent. 

Potassium  ...........................................  28.0  -40  .  o  per  cent. 

Sodium  ..............................................  0.5  -  1.9  per  cent. 

Magnesium  ..........................................  4.0  -  8.1  per  cent. 

Silicic  acid  ...........................................  o.o-  1.6  per  cent. 

Calcium  .............................................  i.o  -  4.5  per  cent. 

Iron  oxide  ...........................................  o.i  -  7-3  per  cent. 

Sulphuric  acid  ........................................  0.6  -  6  .b  per  cent. 

Chlorine  .............................  ................  0.03-  i  .o  per  cent. 

Kappes,  (S.  Anm.  zu  Taf.  I,  5,  52),   Cramer   (Arch.  f.  Hyg.,  28),  De  Schweinitz  and  Dorset 
(Cent.  f.  Bakt.,  23,  993). 

B.  xerosis  B.  prodigiosus,\   B.  tuberculosis,   'Cholera  spirillum, 

per  cent.  per  cent.  per  cent.  per  cent. 

Phosphoric  acid  .......  34.0  36.0  55.2  10.0-45.0 

Potassium  ............  n.o  n.o  6.4  4.0-6.0 

.Sodium  ...............  24.0  28.0  13.6  27.0-34.0 

Magnesium  ...........  6.0  7.0  1  1  .  6  0.1-0.6 

Calcium  ..............  3.0  4.0  12.6  0.3-1.3 

Silicic  acid  ............  0.5  0.5  0.6 

Sulphuric  acid  .........  ....  ....  o.o  r.  0-8.0 

Chlorine  ...............  0.6  5.0  o.o  5.  0-44  .  o 


IQ4  PHYSIOLOGY   OF  MICROORGANISMS 

sodium  occupy  very  prominent  places;  yet  the  relations  of  these  two 
elements  are  sometimes  reversed.  Calcium  and  the  other  constituents 
are  subject  to  considerable  fluctuation.  If  any  inference  is  to  be  drawn 
from  this  work,  it  must  mean  that  phosphorus  is  a  very  important 
element,  serves  an  essential  role,  and  is  of  consequence  to  protoplasm, 
probably  as  a  basic  constituent.  Potassium,  sodium,  magnesium,  and 
calcium  are  uniformly  constant  ingredients,  are  concerned  in  nutritional 
exchanges  and  may  in  a  limited  manner  be  bound  in  the  structure  of  the 
protoplasmic  molecule. 

The  concentration  of  the  culture-medium  and  brine  solutions  are 
known  to  influence  the  amount  of  ash-content  of  microorganisms. 
Cramer,*  using  a  i  per  cent,  sodium  carbonate  bouillon,  a  4  per  cent, 
sodium  phosphate  bouillon  and  a  3  per  cent,  sodium  chloride  bouillon 
obtained  in  the  case  of  Msp.  cholerce  respectively  9.3  per  cent.,  22.3 
per  cent.,  and  25.  9  per  cent,  ash  (dry  weight). 

Other  substances  are  found  present  in  microbial  cells.  These  should 
be  referred  to  here  although  more  extensive  consideration  will  be  given 
some  of  them  later. 

Enzymes  are  found  in  all  microbial  cells.  They  are  agents  employed 
in  metabolism  and  in  the  preparation  of  food  for  incorporation  in  the 
body  of  the  cell  and  incidentally  produce  changes  which  result  in 
products  of  fermentation  as  alcohol.  They  act  very  specifically  inas- 
much as  a  particular  enzyme  is  needed  for  every  substance  changed 
as  cane  sugar,  malt  sugar,  starch,  protein,  fat,  etc.  They  cause  change 
apparently  without  altering  their  nature.  They  are  influenced  by 
many  conditions  of  temperature,  reaction,  accumulated  products,  etc. 
An  organism  is  capable  of  secreting  or  containing  within  its  protoplasm 
several  enzymes,  each  being  produced  only  when  the  cell  is  specifically 
stimulated. 

Toxins  much  like  enzymes  may  be  found  within  the  cell  substance 
or  in  the  medium  in  which  the  microorganism  may  be  growing.  They 
are  associated  with  disease-production  and  pathogenesis.  Their  force 
as  a  poison  (the  meaning  of  the  word)  is  incomparably  great.  Only  a 
small  number  of  microorganisms  are  able  to  produce  toxins. 

Vitamines  are  substances,  somewhat  intangible,  which  have  been 
found  in  some  microorganisms  and  quite  generally  in  food  substances. 
They  are  seemingly  essential  to  life.  Their  recognition  at  the  present  time 
is  largely  by  solubility  and  physiological  determination  upon  animals. 

"Cramer,  Arch.  f.  Hyg  ,  28,  i. 


DIVISION  II 

NUTRITION  AND  METABOLISM 

i 

INTRODUCTION* 

The  nutrition  and  metabolism  of  microorganisms  are  based  on  many 
of  the  same  principles  which  regulate  animal  and  plant  metabolism; 
in  many  ways  microorganisms  are  more  closely  related  to  animals  than 
to  plants,  if  viewed  from  the  standpoint  of  their  food,  their  mode  of 
digestion,  and  their  general  physiological  nature.  Aside  from  the 
many  specific  physiological  processes  peculiar  to  microbial  life  as 
in  the  case  of  life  without  oxygen  (anaerobiosis)  and  in  the  ability  of  some 
species  to  use  free  nitrogen  gas,  the  functioning  of  microorganisms 
accords  with  the  cellular  metabolism  and  nutritive  principles  of 
the  more  highly  developed  organisms.  Since  it  will  be  desirable 
frequently  to  refer  to  plant  and  animal  nutrition  in  the  course  of 
this  discussion,  these  principles,  therefore,  are  briefly  discussed  in 
the  following  paragraphs. 

Green  plants  feed  only  on  inorganic  substances.  They  assimilate 
carbon  dioxide  (COz)  from  the  air  which  unites  with  water,  nitrates, 
potassium,  calcium,  and  other  salts  of  the  soil  and  form  the  body  sub- 
stances of  the  plant.  The  cellulose,  starch,  sugar,  protein  and  all  other 
compounds  constituting  the  plant  cells  are  produced  from  these  simple 
inorganic  substances.  Animals  feed  upon  animals  and  plants.  Unlike 
plants  they  utilize  the  oxygen  of  the  air  and  give  off  carbon  dioxide 
(CO2).  Out  of  these  materials,  together  with  water,  life  is  sustained. 
Although  in  details  animals,  plants  and  microorganisms  differ  quite 
widely,  the  general  laws  of  nutrition  and  metabolism  are  very  similar. 

The  methods  by  which  microorganisms  secure  their  food  vary. 
Molds  take  up  their  food  through  the  mycelium  after  it  has  been  pre- 
pared by  the  action  of  digestive  agents,  enzymes,  secreted  by  the  cells. 
If  the  food  be  suitable  for  the  life  of  the  cell  without  change,  of  course, 
these  digestive  agents  are  not  needed.  When  properly  altered,  such 


*  Prepared  by  Otto  Rahn.     Revised  by  Editor. 

195 


1 96  NUTRITION   AND    METABOLISM 

compounds  enter  as  are  permitted  by  the  cell-wall  and  protoplasm  by 
means  of  osmotic  pressure.  They  then  diffuse  throughout  the  proto- 
plasm of  the  cell.  Other  digestive  agents  within  the  cell  make  the  food 
assimilable.  In  molds  the  food  may  apparently  pass  along  the  myce- 
lium or  hyphae,  in  other  words  be  transmitted  for  some  distance  through 
the  organism.  In  the  case  of  the  yeast  cell  and  bacteria  the  process  is 
very  similar  but  the  transmission  of  nutritive  material  beyond  a  single 
cell  is  not  known  to  take  place  and  perhaps  there  is  no  need  for  it. 
Whether  food  is  conveyed  from  one  cell  to  another  in  colonies  has  not 
been  determined  so  far  as  the  writer  knows. 


Food 

Waste 
Secretions 

Water 


FIG.  no. — Illustrating  cell  activities. 

Waste  products  resulting  from  the  metabolism  of  protoplasm  leave 
the  cell  through  the  cell- wall,  also  by  means  of  osmosis,  and  this  process 
appears  to  be  the  same  for  the  ingestion  of  food  as  for  the  egestion  of 
waste  products. 

Some  microorganisms  live  upon  dead  matter,  some  upon  living 
matter  and  some  may  make  use  of  either.  The  greater  portion,  by  far, 
require  or  prefer  organic  substances.  When  organisms,  as  protozoa, 
feed  upon  living  organisms  they  are  said  to  be  holozoic  in  their  mode  of 
life,  in  other  words  they  follow  closely  the  methods  employed  by  ani- 
mals. Then  there  are  those  protozoal  organisms  which  simulate  plants 
in  their  manner  of  nourishment.  These  are  called  holophytic.  This 
latter  class  is  associated  with  the  formation  of  chlorophyll-bodies  within 
their  structure.  There  are  those  organisms,  too,  which  consume . 
organic  matter  which  is  rendered  suitable  by  nature  or  decay,  called 
saprozoic  or  saprophyte,  depending  upon  whether  the  organism  is 
designated  as  animal  or  plant.  Whenever  organisms  require  living 
tissues  to  sustain  life,  in  the  form  of  a  host,  they  are  called  parasitic. 


INTRODUCTION 


197 


Many  of  these  microorganisms  absorb  their  nutrition  directly  from  the 
fluids  of  the  tissues  while  others,  amoebae,  are  able  to  devour  cells. 

Protozoa  are  very  much  like  all  microorganisms  in  their  manner  of 
living  but  there  are  details  which  belong  to  them  as  a  class  and  should 
be  pointed  out  specifically. 


7, 


;.  i ii. — A,  Amoeba  proteus;  Na,  a  food  particle;  Cv,  contractile  vacuole;  A7,  nucleus. 

(After  Doflein.) 


"The  ingestion  of  food  is  accomplished  in  some  protozoa  by 
pseudopodia;  the  protozoon  simply  flows  around  and  so  encloses  a  food 
particle  (Fig.  in).  In  the  same  way,  these  protozoa  flow  away  from 
waste  particles  which  are  to  be  eliminated.  Other  protozoa  have  defi- 
nite mouth  areas  for  the  ingestion  of  food,  and  definite  anal  areas  for 
the  discharge  of  residual  material.  Those  protozoa  which  ingest  solid 
food,  digest  it  within  gastric  vacuoles  by  the  aid  of  enzymes  and  of 
acids,  just  as  is  the  case  in  many-celled  animals.  The  most  important 
of  the  disease-producing  protozoa  live  within  nutrient  fluids,  for  ex- 
ample the  blood,  and  they  obtain  their  nourishment  from  the  fluid  in 

*  Prepared  by  J.  L.  Todd. 


198  NUTRITION  AND   METABOLISM 

which  they  live,  by  osmosis;  consequently,  they  have  no  definite  mouth 
area,  nor  gastric  vacuoles. 

*"Some  of  the  protozoa,  for  example,  some  amoebae  and  ciliata,  pos- 
sess contractile  vacuoles.  A  contractile  vacuole  is  a  clear  cavity  which 
appears  in  the  cytoplasm,  grows  slowly,  empties  itself  by  a  rapid  con- 
traction of  the  fluid  which  has  drained  into  it  and  forms  again.  The 
fluid  which  it  ejects  contains  the  soluble  waste  products  resulting  from 
the  metabolism  of  the  protozoon.  One  function  of  the  contractile  vacu- 
oles is,  therefore,  excretion;  in  some  protozoa,  they  are  probably  also 
concerned  with  respiration.  Contractile  vacuoles  are  usually  absent 
in  protozoa  which  are  parasitic  within  other  animals. 

*"  The  process  of  respiration  in  the  protozoa  is  in  general  similar  to 
that  of  higher  animals.  Most  of  them  require  oxygen  and  eliminate 
carbon  dioxide.  The  contractile  vacuole  which  is  found  in  certain 
forms  is  believed  to  have  a  respiratory  function.  Respiration  may 
consist  of  the  liberation  of  energy  through  oxidation  or  through  the 
breaking  down  of  complex  molecules.  In  organisms  of  an  anaerobic 
habit  the  respiration  is  probably  through  internal  molecular  changes 
affecting  material  stored  in  the  cytoplasm. 

*"ln  addition  to  the  expulsion  of  solid  undigested  material  from 
the  cytoplasm  there  is  evidence  that  waste  products  other  than  CO2 
are  excreted  by  contractile  vacuoles.  Many  organisms  also  secrete 
material  either  of  the  nature  of  chitinous  membranes  on  their  surface 
or  metabolic  products  in  the  form  of  granules,  etc.,  within  their  bodies. 

*"  Derangement  of  function  may  be  produced  associated  with  it 
are  visible  degenerative  changes.  It  has  also  been  found  that  certain 
protozoa  have  the  ability  to  recover  from  injury  and  to  regenerate  lost 
parts." 

*  Prepared  by  J .  L.  Todd. 


CHAPTER  I 
ENERGY    REQUIREMENTS    IN    CELLULAR     NUTRITION* 

The  formation  of  organic  compounds  from  inorganic  compounds 
requires  a  certain  amount  of  energy.  If  a  certain  quantity  of  sugar  is 
burned  to  carbon  dioxide  (CO2)  and  to  water  (H2O),  a  certain  amount  of 
energy  is  liberated  in  the  form  of  heat.  The  heat  given  off  in  this  case 
is  also  a  distinct  product  of  combustion.  This  heat  is  always  obtained 
in  the  same  amount  regardless  of  the  method  chosen  in  burning  the 
sugar.  It  has  been  definitely  determined  to  be  674  calories  for  i  g. 
molecule  (180  g.)  of  sugar.  The  complete  equation  of  sugar  combus- 
tion is  therefore  written 

C6H12O6  +  120  =  6CO2  +  6H2O  +  674  Cal. 

Consequently  the  same  amount  of  energy  will  be  needed  to  produce 
sugar  from  carbon  dioxide  and  water;  for  the  law  of  the  conservation 
of  energy  requires  that,  if  a  certain  process  liberates  a  certain  quantity 
of  energy,  the  reverse  process  will  require  the  same  quantity  of  energy. 
Green  plants  get  their  energy  from  the  sunlight;  exactly  the  opposite 
proceeds  in  the  equation  which  should  read  from  right  to  left;  CC>2 
and  H2O  are  absorbed  by  the  plant  resulting  in  the  formation  of  sugar. 
But  it  is  evident  from  the  equation  that  C02  and  H2O  are  not  sufficient 
to  produce  sugar  since  it  takes  674  calories  of  heat  in  addition.  The 
radiant  energy  of  light  is  transformed  by  the  chlorophyl  granules  of  the 
plant  leaves  into  chemical  energy  which  causes  the  formation  of  organic 
compounds  from  the  simple  inorganic  or  mineral  matter.  Chlorophyl 
is  the  green  coloring  substance  of  plants,  and  only  green  plants  can  use 
the  energy  of  sunlight  for  their  growth. 

The  growth  of  green  plants  is  a  storing  of  the  energy  of  light  in  the 
form  of  organic  matter;  their  metabolism  is  largely  synthetic,  i.e., 
building  up.  Plants  without  chlorophyl,  however,  like  mushrooms, 
molds,  yeasts  and  bacteria,  have  to  provide  for  their  energy  by  some 
other  means. 

•  Prepared  by  Otto  Rahn. 

199 


200  NUTRITION    AND    METABOLISM 

Animals  construct  their  bodies  mainly  of  organic  matter.  Their 
body  substances  as  protein,  fat,  etc.,  are  derived  from  the  protein, 
fat,  cellulose,  etc.,  of  plants  or  of  animals.  Nevertheless,  a  certain 
amount  of  energy  is  required  in  this  assimilation  process,  since  the 
animal  protein  and  fat  are  somewhat  different  from  the  plant  protein 
and  fat.  Consequently,  complex  chemical  changes  and  rearrangements, 
which  require  some  energy,  are  necessary  for  growth.  Energy  is  also 
lost  by  radiation  of  heat  and  by  locomotion.  Animals,  being  entirely 
unable  to  use  the  sunlight  as  a  source  of  energy,  obtain  their  energy 
from  the  digestion  of  organic  food.  The  larger  part  of  this  food  is 
oxidized  completely;  this  part  provides  the  energy.  The  smaller 
part  of  the  food  is  used  for  building  the  tissues  of  the  body;  it  becomes 
part  of  the  animal  itself.  Animal  metabolism  is  largely  analytic,  i.e., 
destructive  although  a  limited  amount  of  energy  is  required  for  the 
chemical  changes  and  molecular  rearrangements  which  are  essential 
to  animal  tissue  formation — a  synthetic  process.  Accordingly  more 
organic  matter  is  decomposed  than  is  formed.  Often  the  same  sub- 
stance can  serve  both  purposes ;  the  meat  eaten  by  a  dog  furnishes  to 
it  energy  as  well  as  material  for  growth.  In  othe  r  cases,  certain  food 
compounds  execute  only  one  function  and  not  the  other.  This  dis- 
tinction between  food  for  energy  and  food  for  growth  must  also  enter 
into  the  interpretation  of  microbial  metabolism. 

It  might  appear  from  this  discussion  that  energy  is  needed  only  by 
growing  cells,  as  the  full-grown  cells  do  not  increase  in  size  or  weight 
or  number.  They  also  need  energy,  for  in  all  living  cells,  there  is 
noticed  a  continuous  breaking  down  (katabolism)  and  rebuilding 
(anabolism)  of  the  cell  constituents.  This  process  is  commonly  called 
metabolism.  The  katabolic  processes  (the  breaking  down)  in  a  cell 
will  continue  even  if  the  cell  receives  no  food.  The  cell  loses  in  weight 
and  the  starvation  which  follows  will  ultimately  result  in  the  death  of 
the  cell.  All  living  cells  require  food  for  the  maintenance  of  life. 

In  the  first  part  of  this  book,  microorganisms  have  been  divided 
into  plants  and  animals,  but  attention  has  been  called  in  various  places 
to  the  fact  that  it  is  often  hard  to  determine  whether  the  plant  char- 
acters or  the  animal  characters  prevail.  This  holds  true  not  only 
with  the  morphology,  but  also  with  the  physiology  of  microorganisms. 
Since  none  of  the  plants  discussed  in  this  text-book  possesses  chlorophyl, 
none  of  them  can  use  light  as  a  source  of  energy,  therefore  they  depend 


ENERGY   REQUIREMENTS    IN    CELLULAR   NUTRITION  2OI 

entirely  upon  chemical  energy  obtained  by  the  digestion  of  food.  This 
means  that  they  require  organic  food  almost  entirely,  since  inorganic 
food  furnishes  energy  only  in  exceptional  cases.  In  this  respect  they 
resemble  the  animals  very  much. 

The  source  of  energy  in  microbial  life  is  always  of  chemical  origin. 
The  simplest  processes  are  the  oxidations,  and  simplest  among  these 
the  inorganic  oxidations.  A  number  of  different  types  feeding  ex- 
clusively on  minerals  has  been  discovered  during  the  last  twenty  years, 
and  some  of  them  are  of  great  economic  importance.  They  resemble 
plants  in  as  far  as  they  build  their  cells  exclusively  from  carbon 
dioxide,  nitrates  and  ash.  The  food  used  for  building  material  is 
quite  different  from  the  food  used  for  the  provision  of  energy. 

Two  typical  examples  are  the  nitrifying  organisms  in  soil  which 
oxidize  ammonia  to  nitrates.  This  process,  according  to  Winogradski, 
is  divided  distinctly  into  two  phases:  the  Nitrosomonas  oxidizes  the 
ammonia  to  nitrous  acid, 

NH3  +  30  =  HNO2  +  H2O  +  78.8  Cal. 
and  the  Nitromonas  oxidizes  the  nitrous  acid  to  nitric  acid, 
HNO2  +  O  =  HNO3  +  18.3  Cal. 

These  oxidation  processes  yield  a  certain  amount  of  energy  which 
enables  the  bacteria  to  build  their  cells  from  carbon  dioxide,  ammonia, 
and  certain  mineral  salts.  Without  ammonia  or  without  nitrous  acid, 
respectively,  these  bacteria  cannot  grow  for  lack  of  energy;  they  would 
be  like  a  plant  without  light.  It  is  evident  in  this  case  that  the  food  for 
energy  is  also  used  to  some  extent  as  food  for  growth.  The  nitrogen 
necessary  to  the  bacteria  is  supplied  by  the  ammonia  or  the  nitrous  acid. 
As  an  example  distinguishing  strictly  between  the  food  for  growth 
and  the  food  for  energy  may  be  mentioned  the  hyposulphite  bacterium 
studied  by  Nathanson.  This  organism  oxidizes  hyposulphites  to  sul- 
phates and  sulphur,  largely  following  the  formula 

Na2S2O3  +  O  =  Na2S04  +  S  +  x  Cal. 

Hyposulphite  Sulphate      Sulphur 

Besides,  some  more  complex  compounds,  like  sodium  tetrathionate 
(Na^Oe),  are  formed.  The  bacterium  builds  its  cells  exclusively  from 
nitrates,  carbon  dioxide,  and  mineral  salts;  organic  food  is  rejected. 
The  hyposulphite  can  hardly  be  used  for  the  construction  of  the  cell, 
and  must  be  considered  entirely  a  food  for  energy. 


202  NUTRITION  AND   METABOLISM 

This  distinction  is  not  confined  to  mineral  decomposition  only. 
The  urea  bacteria  get  their  energy  from  the  decomposition  of  urea  into 
ammonium  carbonate  which  is  hydrolysis. 

(NH2)2CO  +  2H2O  =  (NH4)2CO3  +  14.3  Cal. 

Urea  Ammonium 

carbonate 

But  the  urea  and  mineral  salts  are  not  sufficient  for  the  development  of 
the  urea  bacteria.  They  cannot  use  urea  as  a  material  for  building  the 
cells,  and  they  cannot  use  carbon  dioxide  or  carbonates;  they  cannot 
grow  unless  a  suitable  material  for  cell  construction  is  added.  Sohngen 
demonstrated  that  a  few  milligrams  of  malic  acid  favor  a  good  develop- 
ment of  the  bacteria.  The  malic  acid  is  used  entirely  for  the  forma- 
tion of  cell  substances.  The  energy  for  this  formation  came  from  the 
urea  fermentation.  This  example  shows  clearly  the  different  require- 
ments for  cell  growth  and  for  the  energy  supply. 

With  the  urea  fermentation,  we  have  changed  not  only  from  inor- 
ganic to  organic  food,  but  also  from  oxidation  processes  to  other 
decompositions. 

Microorganisms  differ  from  the  higher  animals  by  their  less  complete 
metabolism.  The  food  in  the  animal,  if  digested  at  all,  is  oxidized  as  a 
rule  to  the  final  products  of  combustion,  CO2  and  H2O,  the  only  excep- 
tion being  the  nitrogen  which  leaves  the  body  still  in  organic  combina- 
tion as  urea.  With  bacteria,  yeasts  and  molds,  this  is  not  always  the 
case.  Though  some  of  these  organisms  will  bring  about  complete  oxida- 
tion of  the  food  we  find  more  commonly  incomplete  oxidations  or 
changes  which  require  no  oxygen  at  all,  but  still  yield  energy  to  the  cell. 
The  biochemical  side  of  these  changes  of  which  the  alcoholic  fermenta- 
tion is  the  best  known  will  be  discussed  in  the  chapter  on  oxygen 
requirements. 


CHAPTER  II 
MECHANISM  OF  METABOLISM* 

GENERAL  THEORY  OF  METABOLISM 

ANABOLISM,  KATABOLISM,  METABOLISM. — It  has  been  stated  that 
microorganisms  need  food  for  at  least  two  different  purposes :  building 
material  and  building  energy.  They  may  need  it  for  other  purposes 
also,  e.g.,  for  motion.  The  sum  of  all  changes  which  the  food  undergoes 
in  the  body,  including  the  deterioration  of  the  cells,  is  called  metabol- 
ism. Metabolism  consists  of  several  separate  functions:  One  of 
them  is  the  construction  of  new  cells,  or  parts  of  cells,  called  anabol- 
ism,  another  the  deterioration  of  cells,  called  katabolism,  and  the  most 
important  quantitatively  is  the  fermentation  or  respiration.  The 
fermentation  or  respiration  processes  are  fairly  well  understood;  many 
of  them  can  be  produced  in  the  chemical  laboratory  without  micro- 
organisms. Katabolism  is  the  sum  of  many  processes  some  of  which 
are  well  understood  while  others  are  still  unknown.  The  synthetic, 
anabolic  processes  of  the  cell,  however,  are  almost  entirely  unknown, 
and  we  can  only  speculate  regarding  the  various  means  by  which  the 
cell  grows.  The  explanations  of  the  different  cell  activities  began, 
as  in  most  other  fields  of  theoretical  microbiology,  with  a  close  analogy 
with  animal  and  plant  metabolism,  but  owing  to  the  comparative 
simplicity  of  the  microorganisms,  they  led  to  the  establishment  of  new 
facts  and  theories  which  proved  afterward  useful  for  the  understand- 
ing of  the  metabolism  of  the  more  complex  organisms  where  the  multi- 
plicity of  facts  prevented  a  clearer  insight  into  the  separate  processes. 

INTRA-  AND  EXTRA-CELLULAR  FERMENTATION 

DECOMPOSITION  OF  INSOLUBLE  FOOD. — Many  microorganisms  feed 
upon  cellulose,  starch,  fat,  gelatin,  keratin  and  other  insoluble  com- 
pounds. Microorganisms,  with  the  exception  of  some  protozoa, 

*  Prepared  by  Otto  Rahn. 

203 


204  NUTRITION    AND    METABOLISM 

depend  upon  soluble  food  since  they  have  no  means  of  incorporating 
insoluble  compounds  into  their  protoplasm.  The  protoplasm,  however, 
must  be  considered  the  center  of  metabolism,  and  the  digestion  of  food 
and  the  formation  of  energy  must  take  place  in  the  protoplasm  if  the 
cell  is  to  profit  by  it.  Since  the  food  cannot  diffuse  into  the  cell,  and 
the  protoplasm  does  not  diffuse  out,  the  food  must  be  dissolved.  This 
is  accomplished  by  the  cell  itself  by  secreting  certain  agents  with 
peculiar  qualities.  These  agents,  the  so-called  enzymes,  act  upon  the 
insoluble  foods,  changing  them  into  soluble  compounds  which  then  can 
diffuse  into  the  cell  where  they  are  digested  or  fermented.  The  final 
digestion  or  fermentation  of  thetfood  must  take  place  within  the  cell. 
Energy  production  outside  the  cell  serves  the  same  purpose  as  a  stove 
outside  the  house.  The  dissolution  of  insoluble  compounds  by  cell 
secretions  must  be  considered  a  preparatory  process  which  has  no  direct 
relation  to  intra-cellular  food  digestion  or  fermentation.  Enzymes  are 
not  produced  by  microbial  cells  exclusively.  All  living  cells  produce 
enzymes.  They  were  known  before  the  science  of  microbiology  had 
been  established.  In  fact,  microbial  activity  was  considered  for  a 
long  time  as  an  enzymic  chemical  process.  Enzymes  in  the  animal 
and  plant  body  serve  largely  the  purpose  of  metabolic  changes.  In 
the  animal  body,  many  enzymes  help  to  dissolve  the  insoluble  food 
which  cannot  pass  from  the  alimentary  canal  into  the  body  except  by 
diffusion  through  the  mucous  membrane.  There  is  diastase  in  the 
saliva  which  acts  upon  starch,  there  is  pepsin  in  the  stomach  and 
trypsin  in  the  intestine,  both  dissolving  protein  bodies;  there  is  ereptase 
for  the  peptones,  lipase  for  the  fat,  inwrtase  for  the  saccharose,  and 
many  other  enzymes.  The  object  of  all  these  enzymes  is  apparently 
to  prepare  the  food  for  passing  through  the  membrane  into  the  proto- 
plasm of  the  cells,  where  the  final  changes  which  liberate  energy  take 
place.  The  same  processes  occur  with  microorganisms  but  in  a  more 
simple  manner.  Surrounded  by  a  liquid  medium,  they  secrete  enzymes; 
these  dissolve  certain  insoluble  foods  which  then  diffuse  through  the 
cell  wall  to  be  decomposed  further. 

The  food-preparing  processes  are  all  supposed  to  be  simple  hydrolytic 
processes.  For  some  of  these  changes  the  chemical  equations  are  well 
known.  The  hydrolyzation  of  starch  to  maltose  by  means  of  diastase  is 
represented  by  the  equation 

2(C6H10O5)n  +  nH2O   = 


MECHANISM    OF    METABOLISM  205 

The  splitting  up  of  a  fat  molecule  into  glycerin  and  fatty  acid  is  also  a 
well-known  process 

C3H5(C18H3502)3  +  3H20  =  C3H5(OH)3  +  3Ci8H36O2. 

Tristearin  Glycerin  Stearic   acid 

Proteolysis  is  not  so  well  known  and  the  general  supposition  that 
the  first  stages  of  protein  degradation  are  hydrolytic  is  largely  based 
upon  analogies.  Some  of  these  enzymes  which  are  secreted  by  the 
microbial  cells  act  upon  soluble  compounds.  Iniertase  decomposes 
saccharose  into  dextrose  and  levulose: 

C12H22On  +  H,0  =  C6H1206  +  C6H1206. 

Other  disaccharides  are  hydrolyzed  in  the  same  way  by  other  enzymes; 
glucosides  are  decomposed  by  emulsin;  soluble  proteins  are  changed  to 
peptones.  It  is  not  necessary  that  the  enzymes  act  upon  the  soluble 
compounds  outside  the  cell  since  these  compounds  can  diffuse  into  the 
cell;  these  enzymes  are  found  only  occasionally  within  the  cell.  It 
may  be  said,  however,  that  the  smaller  molecules  of  the  products  of 
enzymic  action  diffuse  more  readily  than  the  larger  molecules  of  the 
original  food  compound. 

PROPERTIES  OF  ENZYMES. — These  secretions  of  cells  are  treated  in  a 
group  by  themselves  because  they  differ  distinctly  in  many  respects 
from  any  other  chemical  substance.  Probably  the  most  notable  differ- 
ence may  be  discovered  in  the  fact  that  their  action  does  not  follow  the 
law  of  mass  action  which  supposes  that  all  substances  reacting  upon 
each  other  diminish  in  quantity.  Rennet  will  coagulate  many  hundred 
times  its  weight  of  casein,  and  still  the  whey  will  contain  rennet.  Con- 
sidering that  part  of  the  rennet  is  physically  absorbed  by  the  coagulum, 
the  amount  of  rennet  is  found  to  be  the  same  as  before,  though  it  has 
changed  a  comparatively  enormous  quantity  of  casein.  The  same  is 
true  with  other  enzymes.  The  enzyme  is  not  destroyed  by  acting 
upon  other  substances.  This  exceptional  quality  furnishes  a  reason  for 
treating  enzymes  as  a  separate  group  or  apart  from  other  chemical 
substances.  But  there  are  still  other  qualities  which  distinctly  separate 
them  from  the  well-known  chemical  bodies,  and  show  at  the  same  time 
their  relation  to  proteins  and  toxins  (page  248).  One  of  these  is 
their  sensibility  to  such  outside  influences  as  will  destroy  life.  Enzymes 
are  inactivated  by  exposure  to  temperatures  above  50°  to  80°,  and 


2O6  NUTRITION   AND    METABOLISM 

can,  like  coagulated  albumin,  by  no  means  be  brought  back  to  their 
original  state.  This  temperature  is  very  near  the  coagulating  tempera- 
ture of  albumin.  It  is  believed  from  this  resemblance  that  enzymes 
are  of  an  albuminous  nature.  Another  similarity  is  the  fact  that  both 
enzymes  and  albumins  are  precipitated  by  concentrated  salt  solutions. 
Enzymes  can  further  be  inactivated  by  poisons.  The  same  sub- 
stances which  kill  living  cells,  like  formaldehyde,  hydrocyanic  acid, 
mercuric  chloride,  phenol,  will  also  inactivate  enzymes,  though  usually 
stronger  solutions  are  required  for  the  destruction  of  the  enzyme  than 
for  killing  the  cell.  It  is  the  same  with  heat;  a  higher  temperature  is 
generally  required  to  destroy  the  enzyme  than  to  kill  the  cell  which 
secreted  it.  Light  will  also  affect  enzymes  considerably.  The  great 
similarity  of  enzymes  and  microorganisms  in  these  respects,  the  simi- 
larity of  their  reactions  and  the  extreme  minuteness  of  the  bacteria 
render  it  explicable  why  the  chemists  of  eighty  years  ago  could  not 
determine  the  difference  between  microorganisms  and  enzymes,  and 
called  them  both  "ferments." 

With  the  toxins,  the  enzymes  have  in  common  the  great  sensibility 
to  heat,  light,  and  chemicals.  Both  of  these  groups  are  resistant  to 
drying  to  a  limited  extent.  So  far  as  body  reactions  are  concerned  these 
two  groups  seem  to  belong  to  one  physiological  group  of  compounds. 
When  toxins  are  injected,  the  body  responds  by  the  production  of  anti- 
toxins which  inactivate  the  toxin.  In  the  same  way  the  body  responds 
to  enzymes  by  the  production  of  anti-enzymes  which  prevent  the  action 
of  the  enzymes.  It  may  be  mentioned  that  against  protein  compounds, 
precipitins  are  produced  by  the  body  which  precipitate  only  that  protein 
which  was  injected.  This  " specific"  action  is  also  true  with  toxins  and 
enzymes.  The  anti-body  will  inactivate  only  the  specific  kind  of  toxin 
or  enzyme  that  was  injected. 

What  an  enzyme  really  is  cannot  be  defined.  An  enzyme  is  known 
only  by  its  reactions.  Many  chemists  have  tried  to  prepare  pure  en- 
zymes by  continuously  dissolving  and  precipitating,  by  dialyzing  and 
other  means,  but  there  are  two  great  difficulties  existing;  there  is  no  test 
for  the  purity  of  enzymes,  and  they  lose  in  activity  if  treated  with 
chemicals.  The  more  they  are  freed  from  the  protein  bodies  which 
always  accompany  them,  the  more  sensitive  they  are  to  injurious  in- 
fluences. Mineral  salts  seem  essential  for  their  action,  because  con- 


MECHANISM   OF   METABOLISM  207 

tinued  dialyzing  weakens  the  activity  which  can  be  restored  only  by 
adding  salts. 

ENZYMES  OF  FERMENTATION. — It  has  been  demonstrated  in  the 
above  paragraph  that  food  is  prepared  for  digestion  or  fermentation  by 
enzymes.  The  final  decomposition,  the  process  which  yields  the  energy 
for  cell  life,  must  take  place  within  the  cell. 

The  difference  in  importance  of  food  preparation  and  fermenta- 
tion may  be  illustrated  by  the  example  of  Rhizopus  oryza.  This 
mold  attacks  starch,  changes  it,  by  means  of  diastase,  to  maltose, 
the  maltose  to  dextrose,  dextrose  to  alcohol  and  carbon  dioxide.  The 
mold  grows  well  in  a  starch  medium,  without  sugar;  it  grows  equally 
well  in  maltose,  and  equally  well,  or  better,  in  dextrose;  it  does  not 
grow  at  all  with  alcohol  and  carbon  dioxide.  The  last  change,  dex- 
trose to  alcohol,  is  absolutely  necessary  for  this  organism;  it  is  the 
source  of  its  life;  the  others  are  incidental  processes,  not  absolutely 
necessary  under  all  circumstances,  in  fact  greatly  suppressed  if  dextrose 
is  given  together  with  starch.  The  fermentation  must  take  place  in 
the  cell;  the  preparation  of  food  may  take  place  in  the  cell  or  outside; 
it  is  not  essential  where  it  happens. 

The  investigations  of  recent  years  have  demonstrated  that  fermenta- 
tions also  are  caused  by  enzymes.  It  has  been  proved  beyond  doubt 
that  in  the  alcoholic,  lactic,  acetic  and  urea  fermentations  the  fermen- 
tation process  may  continue  after  the  death  of  the  fermenting  cells. 
In  the  case  of  alcoholic  fermentation,  the  fermenting  agent  was 
separated  first  by  Buchner  from  the  lacerated  cells  and  was 
filtered  through  porcelain  filters  without  losing  its  ability  to  act. 
This  proves  the  enzyme-nature  of  the  fermenting  agent  which,  once 
being  formed,  remains  and  acts  independent  of  the  cell.  These  en- 
zymes are  called  zymases.  They  remain  within  the  cell  as  long  as  it 
is  alive.  They  are  much  more  sensitive  to  injurious  influences  than 
the  above-mentioned  food-preparing  enzymes.  Much  skill  and  pa- 
tience was  required  to  demonstrate  their  independence  of  the  living 
cell.  After  these  enzymes  were  found  in  microorganisms,  similar 
enzymes  were  discovered  in  the  cells  of  higher  plants  and  animals. 
Many  of  the  biochemical  changes  taking  place  in  the  final  dissociation 
of  food  within  the  cell  are  known  to  be  the  result  of  enzymic 
action;  heretofore  these  reactions  were  believed  to  be  a  part  of  the 
life  processes,  inseparable  from  the  living  cell.  Even  some  of  the 


208  NUTRITION    AND    METABOLISM 

oxidations  and  many  reducing  processes  have  been  recognized  as  caused 
by  enzymes,  and  it  is  quite  probable  that  the  whole  process  of  intra- 
cellular  food  decomposition  in  all  organisms  is  accomplished  entirely 
by  means  of  enzymes. 

CLASSIFICATION  OF  ENZYMES 

Since  the  chemical  nature  of  enzymes  and  of  their  action  is  largely 
unknown,  they  can  be  arranged  for  convenience  only  according  to  the 
compounds  they  act  upon.  It  is  possible,  however,  to  distinguish 
between  the  following  four  groups:  Hydrolyzing,  zymatic,  oxidizing, 
reducing  enzymes.  This  definition  is  not  quite  exact,  since  the  urea 
fermenting  enzyme  is  also  a  hydrolyzing  enzyme,  and  the  acetic  fer- 
mentation is  caused  by  an  oxidizing  enzyme.  The  distinction  between 
endo-enzymes  (infra-cellular)  and  exo-enzymes  (secreted)  is  not  exact, 
either,  since  invertase  and  lactase  are  retained  in  the  cells  of  some 
organisms  and  secreted  by  others. 

The  following  classification  is  used    in   the    further  discussions: 

I.  Hydrolytic  Enzymes. 

1.  of  carbohydrates:  cellulase  (cytase),  diastase  (ptyalin,  amylase),  invertase, 
lactase,  maltase. 

2.  of  fats:  lipase  (steapsin). 

3.  of  proteins: 

(a)  proteolytic  (proteases):  pepsin  (peptase),  trypsin  (tryptase),  erep- 
sin  (ereptase). 

(b)  coagulating  (coagulases) :  thrombase,  rennet  (chymosin). 
II.  Zymases. 

1.  of  carbohydrates:  alcoholase,  lactacidase. 

2.  of  other  nitrogen-free  bodies:  vinegar-oxidase. 

3.  of  proteins:  endo- tryptase,  autolytic  enzymes,  amidase,  urease. 

III.  Oxidizing  Enzymes. 
Vinegar-oxidase,  tyrosinase. 

IV.  Reducing  Enzymes. 

Katalase,  reductases  of  nitrates,  sulphur,  sulphites,  telluric  salts,  methylene 
blue,  litmus. 

Several  different  names  have  been  given  to  some  of  the  enzymes; 
these  are  found  in  parenthesis  in  the  above  classification. 

The  general  action  of  enzymes  being  explained  in  the  preceding 
pages,  it  remains  to  describe  more  in  detail  the  different  enzymes  of 
microbial  origin. 


MECHANISM    OF    METABOLISM  2OQ 

HYDROLYTIC  ENZYMES 

ENZYMES  OF  CARBOHYDRATES. — Enzymes  which  decompose  carbo- 
hydrates are  very  commonly  found  in  nature,  because  carbohydrates 
constitute  a  very  extensive  and  common  group  of  organic  matter. 
By  far  the  largest  part  of  the  dry  plant  consists  of  cellulose,  starch 
and  sugar.  To  decompose  them,  enzymes  are  necessary.  The  chem- 
ical reaction  of  these  enzymes  is  hydrolytic;  in  other  words,  the  larger 
molecule  is  broken  into  smaller  ones  by  the  simple  addition  of  water. 
Thus,  the  cellulose-destroying  enzyme,  called  cellulase  or  cytase,  de- 
composes the  cellulose  into  soluble  sugars  after  the  following  formula : 

C6H10O5  +  H2O  =  C6H12O6 

or,  considering  that  the  cellulose  molecule  is  really  many  times 
C6HioO5,  the  formula  will  be  more  accurately  written 

(C6H1005)n  +  nH20  =  nC6H1206 

which  indicates  at  the  same  time  that  one  cellulose  molecule  gives 
many  sugar  molecules. 

Cellulase  is  an  enzyme  which  is  quite  difficult  to  obtain.  Though 
it  must  be  produced  by  all  the  cellulose  destroying  molds  and  bacteria, 
experiments  have  failed  in  some  instances  to  prove  its  presence.  It 
is  found  in  some  wood  destroying  fungi  and  in  some  of  the  bacteria 
causing  the  rot  of  vegetables.  The  organisms  of  certain  plant  diseases 
force  their  way  into  the  cell  by  dissolving  the  cellulose  membrane  by 
an  enzyme,  while  certain  molds  are  able  to  puncture  the  cell  wall 
mechanically. 

Diastase,  or  amylase,  is  the  starch-dissolving  enzyme  which  is  one 
of  the  most  common  enzymes  in  nature.  It  is  found  in  all  green  plants, 
and  it  forms  during  the  sprouting  of  starchy  seeds.  Many  molds 
and  a  few  bacteria  produce  this  enzyme,  while  yeasts  generally  cannot 
decompose  starch  for  lack  of  diastase.  Starch  has  the  same  formula 
as  cellulose,  and  it  is  broken  up  into  soluble  sugars  in  the  same  way. 
Much  attention  has  been  paid  to  this  process  by  the  chemists,  and  it 
is  found  that  the  process  is  a  gradual  one,  giving  first  dextrins,  and 
finally  maltose  (Ci2H22On).  The  hydrolysis  of  starch  expressed  in 
chemical  symbols  may  be  presented  as  follows: 

2(C6H10O6)n  +  nH2O  =  nCi2H22On. 

Starch  Maltose 

14 


210  NUTRITION   AND   METABOLISM 

The  disaccharides  or  double  sugars,  having  the  chemical  formula 
Ci2H22On  are  broken  up  into  single  sugars,  monosaccharides,  by  the 
following  process: 


Ci2H22On  +  H2O  =  C6H12O6  +  C6H12O6. 

The  two  molecules  of  C6Hi2O6  are  different  with  different  sugars. 
If  the  disaccharide  is  saccharose,  the  two  monosaccharide  molecules 
are  dextrose  and  levulose.  Lactose  will  yield  dextrose  and  galactose, 
and  maltose  will  give  two  molecules  of  dextrose.  For  each  of  these 
sugars,  there  is  a  special  enzyme  which  can  hydrolyze  only  its  par- 
ticular sugar  and  none  of  the  others;  like  a  key,  made  for  one  lock, 
it  will  not  open  another  lock.  Maltase  will  split  only  maltose  mole- 
cules, not  lactose,  while  the  lactase  cannot  attack  the  maltose.  In- 
vertase (or  sucrase)  will  decompose  nothing  but  saccharose.  This 
decomposition  of  the  complex  sugars  into  the  simple  sugars  was  be- 
lieved to  be  necessary  because  only  sugars  of  the  type  C6Hi2O8  can 
be  fermented  directly  by  the  fermenting  enzyme  in  the  cell,  be  it  an 
alcoholic  or  lactic  or  gassy  fermentation.  This  explains  why  beer  yeast 
cannot  ferment  lactose;  it  produces  no  lactase,  and  therefore  cannot 
attack  the  lactose  molecules;  they  would  be  easily  attacked,  if  besides 
the  yeast,  some  lactase  were  added.  Certain  lactic  bacteria  cannot 
ferment  saccharose,  because  they  do  not  form  invertase.  Recent 
experiments  have  shown  that  bacteria  exist  which  ferment  lactose 
and  saccharose  but  not  dextrose  or  levulose.  An  explanation  for  this 
cannot  be  given. 

Invertase  is,  like  diastase,  a  very  common  enzyme  in  green  plants. 
It  is  also  produced  by  most  molds  and  yeasts,  and  bacteria.  Maltase 
is  not  quite  so  common,  and  lactase  is  limited  to  a  few  species  of 
microorganisms.  A  few  organisms  are  known  which  do  not  secrete 
these  enzymes  but  retain  them  within  the  cell.  This  is  especially 
true  of  lactase,  but  is  also  known,  in  -a  few  instances,  of  invertase. 
The  enzyme  can  be  obtained  from  the  broken  cells.  Such  enzymes 
are  called  endo-enzymes. 

The  decomposition  of  carbohydrates  has  been  followed  from  the 
most  complex  representatives  to  the  simplest  ones,  the  monosacchar- 
ides. If  these  are  decomposed  further,  the  resulting  product  is  no 
longer  a  carbohydrate.  The  simplest  sugars  are  decomposed  by  zy- 
mases,  inside  the  microbial  cell,  into  compounds  which  are  generally 


MECHANISM    OF   METABOLISM  211 

called  fermentation  products;  these  may  result  from  alcoholic,  lactic, 
butyric  fermentations  or  some  other. 

Emulsin  is  an  enzyme  which  is  able  to  hydrolyze  glucosides.  Gluco- 
sides  occurring  in  plants  are  complex  bodies  which  contain  a  sugar- 
radical.  Emulsin  splits  glucosides  liberating  the  sugar,  usually  dex- 
trose. The  typical  example  for  emulsin  action  is  the  hydrolysis  of 
amygdalin  to  hydrocyanic  acid,  benzaldehyde  and  dextrose. 

C20H27OnN  +  2H2O  =  C6H5COH  +  2C6H12O6  +  HCN. 

Amygdalin  Benzaldehyde  Dextrose  Hydrocyanic  acid 

Emulsin  is  found  in  many  molds  and  bacteria,  and  recently  has 
been  found  in  yeasts.  Glucoside-splitting  enzymes  play  an  important 
r61e  in  the  fermentations  of  coffee-beans,  cocoa,  mustard  and  indigo. 
In  most  of  these  fermentations,  however,  the  emulsin  is  probably  not 
formed  by  microorganisms,  but  by  the  plant,  from  which  the  ferment- 
ing material  is  derived. 

ENZYMES  or  FATS. — All  the  enzymes,  acting  on  fat,  decompose  it 
in  the  same  manner;  the  fat  molecule  takes  up  three  molecules  of  water, 
breaking  up  into  glycerin  and  three  molecules  of  fatty  acid,  as  indicated 
on  page  239.  It  is  possible  that  there  are  several  fat-splitting  enzymes, 
but  the  result  of  the  cleavage  process  is  always  the  same.  The  name 
formerly  assigned  to  enzymes  of  fat  is  steapsin,  but  this  term  is  now 
almost  exclusively  substituted  by  the  more  significant  word  lipasc. 
Occasionally  they  are  called  lipolytic  enzymes  which  expression  is 
analogous  to  the  proteolytic  enzymes;  in  the  same  way,  the  term 
amylolytic  enzyme  is  used  for  diastase. 

ENZYMES  OF  PROTEINS. — The  enzymes  composing  protein  bodies, 
generally  called  proteolytic  enzymes  or  proteases,  have  been  known 
for  nearly  a  century.  Though  the  difficulty  of  analyzing  protein  bodies 
accurately  prevents  an  absolute  knowledge  of  proteolysis,  much  effort 
has  been  made  to  become  acquainted  with  the  very  important  group 
of  enzymes  which  accomplish  the  digestion  of  protein  food.  Naturally 
most  experimenting  has  been  conducted  with  pepsin  and  trypsin 
of  the  animal  body  and  accordingly  these  are  better  understood  than 
others;  only  little  work  has  been  done  with  microbial  enzymes.  There 
is  so  far  as  can  be  determined  little  appreciable  difference  between 
the  proteolytic  enzymes  obtained  from  different  organisms,  whether 
low  or  high  in  the  plant  or  animal  world,  consequently  many  experi- 


212  NUTRITION   AND    METABOLISM 

ences  with  animal  pepsin  and  trypsin  can  be  applied  to  microbial 
enzymes. 

The  specific  chemical  action  of  these  enzymes  is  referable  to  hydro- 
lysis; the  large  protein  molecule  is  broken  up  into  smaller  molecules 
by  addition  of  water.  Various  proteolytic  enzymes  differ  in  the  extent 
of  decomposition.  While  some,  like  pepsin,  produce  mainly  peptones, 
trypsin  is  able  to  split  protein  to  amino-acids  and  even  to  ammonia. 
Mavrojann  is  tested  for  the  intensity  of  gelatin  decomposition  with 
formaldehyde.  The  peptones  of  gelatin  will  solidify  with  formalde- 
hyde while  amino-acids  are  not  affected. 

Proteolytic  enzymes  were  first  divided  into  two  groups:  pepsins, 
which  act  best  in  slightly  acid  solutions,  and  trypsins,  which  act  best 
in  slightly  alkaline  media.  The  names  are  derived  from  pepsin  (peptase) 
the  proteolytic  enzyme  of  the  animal  stomach,  and  from  trypsin  (tryp- 
tase)  which  is  found  in  the  small  intestine  of  animals.  This  classifi- 
cation cannot  be  used  for  the  enzymes  of  microorganisms  because 
there  is  no  definite  line  established  by  the  acidity.  Some  enzymes 
work  in  either  acid  or  alkaline  media  equally  well,  preferring  a  neutral 
reaction.  Enzymes  should  be  classified  according  to  the  substances 
they  act  upon  or  perhaps  according  to  the  nature  of  the  products 
resulting  from  the  fermentation.  This  would  bring  pepsin  and  tryp- 
sin into  one  class,  both  acting  upon  protein  bodies  as  such;  they, 
however,  differ  in  the  intensity  of  action  as  shown  by  their  products, 
the  pepsin  forming  mainly  peptones,  the  trypsin  carrying  on  the 
decomposition  as  far  as  amino-acids  and  traces  of  ammonia.  Another 
class  recently  recognized  is  ereptase  (erepsin)  which  cannot  decom- 
pose protein,  but  readily  attacks  peptones,  decomposing  them  much 
in  the  same  way  as  trypsin.  Pepsin,  trypsin  and  erepsin  do  not 
break  up  amino-compounds. 

The  presence  of  proteolytic  enzymes  in  microorganisms  is  readily 
tested  by  cultivation  on  nutrient  gelatin.  The  proteolytic  enzyme 
secreted  by  the  cells  will  liquefy  the  gelatin.  Generally,  an  organism 
that  liquefies  the  gelatin  will  also  decompose  the  casein  of  milk  and  the 
protein  of  blood  serum.  There  are  some  exceptions,  however,  as  is 
shown  in  the  following  table,  after  Frost  and  McCampbell.  A  + 
sign  means  proteolysis,  a  —  sign  means  no  action. 


MECHANISM    OF    METABOLISM 


2I3 


Milk 
Organism                                                                   Gelatin  Serum 
Coag.    Digest. 

Egg 
album. 

Fibrin 

Bad  anthracis                                                  ~\~ 

+ 

+ 
+ 

+ 

Microspira  comma  +          +          + 
M.  pyogenes  var.  anreus  +          +          + 
Pseudomonas  pyocyanea  +          +          -f- 
B  violaceus.                 .              —                      -f- 

B  mycoidcs                                                      ~\~                      ~\~ 

B  prodigiosus  —          -4-          -4- 

AspCTgillus  nigcT                                                ~\~ 

Aspergillus  oryza  —          +          + 

Apparently  not  all  organisms  which  liquefy  gelatin  are  able  to  de- 
compose egg  albumin;  we  must  conclude  that  the  enzyme  liquefy- 
ing gelatin  is  different  from  the  proteolytic  enzyme  dissolving  egg- 
white. 

COAGULATING  ENZYMES. — The  blood-clotting  enzyme  (throm- 
base)  does  not  occur  in  microorganisms.  Rennet,  however,  is  found 
in  many  species.  Rennet  is  extracted  from  the  stomach  of  calves 
and  pigs  and  used  to  set  the  curd  in  milk  for  cheese  making.  The 
enzyme  acts  upon  the  casein  in  milk,  decomposing  it  into  paracasein 
and  some  soluble  protein.  The  time  of  coagulation  depends  upon 
the  temperature  of  the  milk  and  the  concentration  of  the  rennet. 
This  coagulation  of  milk  is  quite  different  from  the  acid  curd,  where 
the  insoluble  casein  is  precipitated  by  the  acid.  If  enough  acid  is 
added,  the  milk  curdles  immediately;  if  there  is  not  enough  acid, 
there  will  be  no  curd,  not  even  after  a  long  time.  An  acid  curd  can 
be  brought  back  to  the  original  state  by  an  addition  of  alkali,  while 
a  rennet  curd  by  no  means  can  be  changed  back  to  casein.  Rennet- 
forming  bacteria  are  found  in  milk  and  dairy  products,  in  soil  and  other 
habitats.  They  will  coagulate  milk  without  causing  any  appreciable 
increase  of  acidity.  They  all  seem  to  digest  the  curd  after  it  is  formed 
(see  the  above  table).  The  relation  between  proteolytic  and  rennet 
enzymes  will  be  discussed  in  a  later  chapter. 

Rennet  is  sometimes  called  chymosin;  the  Society  of  American 
Bacteriologists  uses  the  German  word  "lab." 


214  NUTRITION  AND   METABOLISM 

ZYMASES 

The  zymases  are  the  agents  which  furnish  the  energy  for  cell  life 
by  causing  fermentative  decompositions.  As  has  been  stated  before, 
the  processes  which  provide  for  energy  must  take  place  inside  of  the 
cell.  Consequently,  all  fermenting  enzymes  are  endo-enzymes.  The 
difference  between  the  soluble  enzymes  and  the  endo-enzymes  is  very 
plainly  shown  in  the  following  table,  giving  the  energy  liberated  by 
the  various  enzymes  by  acting  upon  i  g.  of  substance. 

ENERGY  LIBERATED  FROM  i  G.  OF  SUBSTANCE 

Soluble  Enzymes  Endo-enzymes 

Pepsin,  trypsin o  calories        Lactacidase 80  calories 

Lipase 4  calories        Alcoholase 120  calories 

Maltase,  invertase 10  calories        Urease 230  calories 

Lactase 23  calories        Vinegar-oxidase 2,500  calories 

The  microbial  cell  does  not  lose  much  energy  by  the  activity  of 
the  soluble  enzymes  outside  of  the  cell,  because  their  energy  yield  is 
insignificant. 

The  first  zymase  known  was  urease,  the  enzyme  which  changes 
urea  to  ammonium  carbonate.  The  actual  investigation  of  the 
zymases  did  not  start  until  Buchner  had  demonstrated  that  yeast  can 
be  ground  with  infusorial  earth  until  all  cells  are  lacerated,  and  then 
can  be  pressed  and  the  juice  filtered  without  losing  the  power  of  alco- 
holic fermentation.  Such  fermentation  cannot  be  due  to  anything 
but  a  soluble  compound  of  the  yeast  cell.  Thus  the  alcoholase  was  dis- 
covered. It  was  found  later  that  yeast  may  be  killed  by  alcohol, 
ether  or  acetone  without  losing  its  fermenting  power. 

This  last  method  was  applied  later  to  lactic  bacteria,  and  it  was 
proved  that  the  lactic  acid  is  also  produced  by  an  enzyme,  lactaci- 
dase.  It  is  possible  to  kill  the  lactic  bacteria  so  that  they  do  not 
multiply  but.  still  continue  to  form  acid.  It  seems  quite  probable 
that  other  fermentations  of  carbohydrates,  like  the  butyric  and  the 
gassy  fermentations,  are  really  due  to  enzymes.  It  is  very  difficult 
to  give  the  experimental  proof,  however.  These  enzymes  are  so  un- 
stable that  it  requires  much  experience  to  separate  them  from  the  cell, 
and  it  is  also  quite  difficult  to  obtain  bacteria  in  quantities  large 
enough  for  such  experiments. 


MECHANISM   OF   METABOLISM  215 

The  vinegar  oxidase  is  an  enzyme  which  remains  in  the  cell  of  the 
acetic  bacterium,  oxidizing  alcohol  to  acetic  acid.  Its  independence  of 
the  living  cell  has  been  demonstrated  by  killing  the  cells  with  acetone. 

The  PROTEOLYTIC  ENDO-ENZYMES  of  yeasts,  only,  have  been  studied 
extensively.  That  such  enzymes  exist  is  recognized  by  the  observa- 
tion that  certain  microorganisms  do  not  liquefy  the  gelatin  until 
after  they  are  dead  and  the  proteolytic  enzymes  diffuse  out  through 
the  deteriorating  cell  membranes.  That  yeast  in  the  absence  of 
sugar  loses  in  weight,  and  that  leucin  and  other  cleavage-products  of 
protein  are  formed,  was  the  first  indication  of  a  proteolytic  process  in 
the  yeast  cells.  By  pressing  the  juice  out  of  the  ground  yeast  cells, 
a  liquid  is  obtained  which  liquefies  gelatin,  digests  casein,  albumin  and 
fibrin.  The  living  yeast  cell  does  not  attack  these  compounds,  be- 
cause they  cannot  diffuse  into  the  cell  and  the  enzyme  cannot  diffuse 
out.  The  proteolytic  endo-enzyme  of  yeast  is  called  endo-tryptase. 
Its  object  is  apparently  the  regulation  of  the  protein-content  of  the  cell 
and  perhaps  it  has  some  bearing  on  the  formation  of  cell  plasma. 
The  possible  relation  between  enzymes  and  growth  is  discussed  in  a 
following  sub-chapter. 

If  yeast  is  mixed  with  a  weak  antiseptic  (chloroform,  toluol) 
the  proteolytic  process  takes  place  quite  rapidly.  This  process  is 
called  autolysis  (self-digestion).  Similar  autolytic  enzymes  are  found 
in  other  microorganisms.  Autolysis  is  a  well-known  process  in  the 
higher  animals.  To  this  is  due  the  ripening  of  meat. 

Proteolytic  endo-enzymes  must  be  expected  in  all  microorganisms 
which  depend  upon  protein  as  food  material  only.  These  organisms 
will  secrete  certain  enzymes  which  decompose  the  insoluble  protein 
into  bodies  which  diffuse  easily  into  the  cell.  Here,  proteolytic  endo- 
enzymes  further  decompose  these  products.  Such  an  endo-enzyme  is 
the  amidase  discovered  by  Shibata  in  the  mycelium  of  Aspergillus 
niger  which  forms  ammonia  from  urea,  acetamid,  oxamid,  biuret. 
Endo-erepsin  and  amidase  were  also  found  in  Penicillium  camemberti 
by  Dox. 

Similar  to  these  proteolytic  enzymes  is  the  urease  which  is  formed 
in  large  quantities  in  the  so-called  urea  bacteria,  but  it  is  also  present 
in  the  mycelium  of  some  molds.  An  endo-enzyme,  splitting  hippuric 
acid  into  benzoic  acid  and  glycocoll,  is  found  in  the  mycelium  of  a  few 
molds. 


2l6  NUTRITION    AND    METABOLISM 


OXIDIZING  ENZYMES 

The  most  typical  example  of  an  oxidizing  enzyme  is  the  vinegar- 
oxidase,  because  its  chemical  action  is  well  known.  Most  of  the  oxi- 
dases  known  act  upon  complex  organic  compounds,  changing  them  to 
colored  bodies.  Such  an  oxidase  is  the  tyrosinase  which  forms  a 
black,  insoluble  compound  in  tyrosin  solutions.  It  is  produced  by 
several  bacteria,  especially  by  chromogens,  and  its  application  in  test- 
ing for  small  quantities  of  tyrosin  has  been  suggested.  A  number  of 
oxidases  are  known  to  act  upon  the  leuco-bodies  of  certain  organic  dye- 
compounds,  as  aloin,  guaiac,  phenolphthalein,  and  others.  Hydro- 
chinon  is  oxidized  by  the  dead  cells  of  a  few,  molds.  Strange  seems 
the  oxidation  of  potassium  iodide  to  iodine  by  the  endo-oxidase  of 
a  mold.  Many  other  oxidations  are  supposed  to  be  of  enzymic  nature, 
but  their  independence  of  the  living  cell  has  not  been  proved. 

Many  higher  organisms  are  known  to  contain  oxidases,  the  best 
studied  are  those  of  certain  mushrooms  which  change  the  white  mush- 
room meat  into  a  bluish  or  brownish  color  as  soon  as  it  is  exposed  to 
the  air.  Oxidases  are  very  common  in  most  of  the  tissues  of  higher 
animals. 

REDUCING  ENZYMES 

Among  the  reductases,  one  enzyme  stands  apart  from  all  the  others, 
that  is  the  katalase  or  peroxidase  which  reduces  the  hydrogen  peroxide 
to  water  by  liberation  of  oxygen. 

H2O2  +  katalase  =  H2O  +  O. 

Katalase  is  one  of  the  most  commonly  found  enzymes;  it  is  formed 
by  practically  all  plants  and  all  animals  and  is  contained  by  all  but  a  few 
bacteria.  Among  these  exceptions  is  the  Strept.  lacticus.  The  ab- 
sence of  katalase  in  this  species  has  been  recommended  as  a  diagnos- 
tic test.  It  is  possible  that  this  enzyme  is  necessary  for  intra-cellular 
oxidations. 

A  number  of  other  reductases  are  known.  Nearly  all  of  the  re- 
ductions mentioned  in  the  paragraph  on  the  products  of  mineral 
decomposition  are  proved  to  be  of  enzymic  nature;  these  processes 
will  take  place  after  the  cell  is  killed  by  a  disinfectant  or  is  ground  to 
pieces.  This  can  be  readily  demonstrated  by  lacerating  the  cells 


MECHANISM    OF   METABOLISM  217 

with  quartz  sand.  They  will  then  reduce  nitrates  to  nitrites,  sulphur 
to  hydrogen  sulphide.  The  decolorization  of  litmus,  methylene 
blue,  indigo,  and  other  organic  dyes  is  due  in  microbial  cultures  to 
enzymes  which  are  almost  exclusively  endo-enzymes. 

ENZYMIC  THEORY  OF  KATABOLISM 

Regarding  katabolism  as  the  sum  of  all  destructive  processes  of 
the  living  cell  substance,  i.e.,  of  the  protoplasm,  and  considering  the 
cell  substance  to  be  decomposed  and  renewed  constantly  as  long  as 
the  cell  is  performing  the  normal  functions  of  life,  there  must  be  a  reno- 
vating and  a  destructive  process  continuously  going  on  in  the  proto- 
plasmic molecules.  If  the  food  supply  ceases,  anabolism  ceases  with 
it,  but  it  has  been  demonstrated  that  katabolism  may  continue  just 
the  same  for  some  time.  By  this  method,  the  products  of  katabolism 
can  be  obtained  separate  from  the  products  of  food  digestion  which 
would  obscure  the  results  of  experiment  on  katabolism  in  normally  fed 
cells. 

It  is  difficult  to  determine  to  what  extent  katabolism  is  controlled 
by  endo-enzymes,  the  so-called  autolytic  enzymes,  which  have  been  men- 
tioned in  the  above  paragraph.  Unquestionably,  the  katabolic  processes 
are  similar  to  enzyme  processes,  since  katabolism  is  checked  by  heat 
or  poison  just  like  enzyme  processes. 

ENZYMIC  THEORY  or  ANABOLISM 

ANABOLISM  AND  INTRA-CELLULAR  ENZYMES. — All  changes  dis- 
cussed in  the  previous  chapters  are  processes  in  which  organic  or 
inorganic  compounds  are  broken  up  to  smaller  molecules.  These 
processes  are  exothermic,  i.e.,  liberating  heat  or  energy  in  other  forms. 
The  opposite  is  true  of  the  anabolic  processes  which  build  up  complex 
molecules  from  simple  compounds.  These  synthetic  processes  are 
endothermic,  absorbing  heat  or  other  energy.  Growth  is  the  typical 
manifestation  of  anabolism.  It  is  the  formation  of  new  cells  from  dead 
organic  or  inorganic  matter,  and  it  means  the  formation  of  all  the  com- 
pounds necessary  for  cell  life.  Of  all  the  substances  found  in  the  cell, 
practically  none  are  contained  in  the  food,  and  it  is  wonderful  that 
in  such  a  small  unit  as  a  microbial  cell,  there  are  contained  the  powers 
of  making  protoplasm,  enzymes,  nuclear  bodies,  chromatin  bodies, 
the  substance  of  the  cell  wall  and  probably  many  other  unknown 


2l8  NUTRITION   AND    METABOLISM 

compounds.  All  these  complex  substances  are  generally  made  from 
simple  food  compounds  as  amino-acids,  carbohydrates  and  others. 

These  synthetic  processes  of  the  cell  will,  like  most  endothermic 
processes,  take  place  only  if  energy  is  provided.  This  condition  is 
usually  fulfilled  in  the  living  cell,  due  to  the  fermenting  processes 
going  on  continuously.  There  is  a  strange  interaction  between 
anabolism  and  intra-cellular  fermentation  proceeding  in  the  pro- 
toplasm and  this  linking  together  of  destructive  and  constructive 
reaction  is  the  basis  of  life  processes.  The  life  processes  decompose 
certain  substances,  the  energy  liberated  allows  the  formation  of  proto- 
plasm, which  again  liberates  energy.  Thus  a  continuous  formation  of 
protoplasm  is  secured. 

An  explanation  of  anabolism  based  upon  chemical  experiments  is 
not  possible  at  the  present  time.  In  the  study  of  intra-cellular  destruc- 
tion it  is  possible  to  trace  most  processes  back  to  enzymic  action. 
There  our  knowledge  ceases  because  the  nature  and  mode  of  action 
of  enzymes  is  unknown.  In  the  study  of  anabolism  our  knowledge 
has  not  even  progressed  so  far.  The  most  promising  explanation  at 
present  is  based  upon  the  reversibility  of  enzymic  action. 

REVERSIBILITY  or  ENZYMIC  ACTION 

Chemical  reactions  between  organic  compounds  proceed  quite 
rapidly  at  first,  then  become  slower  and  slower  until  the  reaction 
stops  entirely.  The  reaction  is  not  complete  at  the  time  it  reaches 
an  equilibrium.  If  the  equilibrium  is  disturbed  by  adding  more  of 
the  reagents,  the  process  will  continue.  If,  however,  the  products  of 
reaction  are  added,  the  reverse  process  will  take  place.  Reactions 
between  organic  compounds  can  proceed  either  way,  depending  upon 
the  relative  concentrations  of  the  reacting  substances.  The  standard 
example  is  esterification.  Acetic  acid  plus  alcohol  gives  ester  plus 
water, 


CHsCOOH  +  CH3CH2OH^CH3COOCH2CH3  +  H2O. 

Acetic  acid  Alcohol  Ester 

The  process  goes  to  a  certain  equilibrium  and  stops.  If  ester  is  mixed 
with  water,  it  gives  acid  plus  alcohol,  until  the  same  equilibrium  is 
reached.  If  acid  and  alcohol  are  added  to  a  system  in  equilibrium,  more 
ester  will  be  formed.  If  ester  is  added,  more  alcohol  and  acetic  acid 


MECHANISM  OF  METABOLISM  2IQ 

will  be  formed.  The  same  is  true  with  enzymes,  at  least  with  some 
enzymes.  Maltase  will  decompose  maltose  into  two  molecules  of 
dextrose.  In  a  concentrated  solution  of  dextrose,  however,  maltase 
will  form  maltose,  or  a  similar  sugar,  isomaltose.  Lipase  is  able  to 
produce  fat  from  glycerin  and  fatty  acids.  A  solution  of  albumose 
with  trypsin  or  pepsin  gives  a  precipitate  of  a  body  which  is  more  com- 
plex than  albumose  and  which  gives  the  protein  reactions.  It  is 
believed  by  many  physiologists  that  pepsin  and  rennet  are  the  same 
body.  Under  certain  conditions,  it  has  a  dissolving  power,  under  other 
conditions  it  has  the  power  to  coagulate. 

The  reversibility  of  enzymic  action  has  given  rise  to  much  specula- 
tion about  assimilation  and  growth.  It  seems  reasonable  to  suppose 
that  the  cell  forms  its  protoplasm  from  amino-acids  by  the  reversed 
action  of  proteolytic  enzymes.  In  the  same  way,  cellulose  may  be 
formed  from  dextrose,  fat  from  glycerin  and  fatty  acids.  Nearly  all 
phases  of  growth  can  be  accounted  for  in  this  way.  This  is  nothing  but 
theoretical  speculation,  and  the  only  fact  to  support  it  is  the  reversi- 
bility of  certain  enzymes.  The  conditions  under  which  chemical  reac- 
tions take  place  inside  of  the  cell  are  very  largely  unknown.  There 
are  so  many  processes  going  on  at  the  same  time  that  it  is  absolutely 
impossible  at  the  present  time  to  obtain  a  perfect  understanding  of  all 
these  reactions.  Thus,  our  knowledge  of  growth  is  largely  based 
upon  analogy  and  speculation. 

GENERAL  ENZYMIC  CONSIDERATIONS 

Enzymes  are  produced  only  by  living  cells.  After  they  are  once 
formed,  they  act  like  chemical  compounds,  independent  of  the  cell 
which  produces  them.  Even  the  endo-enzymes  follow  only  the  law  of 
enzyme-action  and  are  not  influenced  by  the  cell  which  contains  them. 
The  enzymes  are  mostly  influenced  by  their  own  products,  and  when 
a  certain  yeast  ceases  to  ferment  sugar  at  the  concentration  of  8.5 
per  cent  of  alcohol,  this  means  that  the  alcoholase  of  this  yeast  cannot 
tolerate  more  than  8.5  per  cent  of  alcohol.  The  inability  of  the  cell 
to  regulate  enzymic  action  may  account  for  the  fact  that  often  a 
culture  produces  an  amount  of  fermentation  products  sufficient  to 
kill  all  cells.  This  is  observed  in  the  lactic,  acetic  and  alcoholic  fer- 
mentations, and,  perhaps,  occurs  in  many  others. 


220  NUTRITION   AND   METABOLISM 

Probably  all  cells  produce  several  enzymes  Microorganisms 
feeding  upon  various  foods  must  form  various  enzymes.  Frequently 
several  enzymes  are  necessary  for  the  decomposition  of  one  com- 
pound. Rhizopus  oryza  uses  three  enzymes  in  order  to  form  alcohol 
from  starch,  first  the  diastase  to  change  starch  to  maltose,  then 
maltase  to  change  maltose  to  dextrose  and  finally  alcoholase 
to  change  dextrose  to  alcohol  and  carbon  dioxide.  The  number  of 
enzymes  formed  by  certain  microorganisms  is  surprising.  Asper- 
gillus  niger  has  the  reputation  of  forming  almost  all  enzymes  which 
have  ever  been  found  in  microorganisms.  Penicillium  camemberti 
produces  (after  Dox)  erepsin,  nuclease,  amidase,  lipase,  emulsin, 
amylase,  inulase,  raffinase,  invertase,  maltase  and  lactase.  It  has 
been  believed  for  a  long  time  that  certain  enzymes  are  regular  products 
of  the  cell  while  others  are  formed  only  if  the  substance  upon  which 
they  act  is  present.  According  to  Dox's  investigations  with  Peni- 
cillium camemberti,  there  is  no  evidence  that  enzymes  not  normally 
formed  by  the  organism  in  demonstrable  quantities  can  be  developed 
by  special  methods  of  nutrition.  The  addition  of  a  particular 
food  compound  does  not  develop  an  entirely  new  enzyme,  but  stimu- 
lates the  production  of  the  corresponding  enzyme  which  is  normally 
formed,  although  in  small  amounts,  under  all  conditions. 


CHAPTER  III 
FOOD  OF  MICROORGANISMS* 

MOISTURE  REQUIREMENT 

Moisture  may  be  called  the  most  important  factor  of  life.  Not 
only  bacteria,  but  every  microscopic  and  macroscopic  being  requires  a 
considerable  amount  of  moisture.  Living  organisms  contain  on  the 
average  between  70  per  cent  and  90  per  cent  of  water,  and  only  10  per 
cent  to  30  per  cent  of  solid  matter.  Microorganisms  which  live 
entirely  submerged  in  liquids  need  water  not  only  within  but  without 
the  cells.  Bacteria,  yeasts,  molds,  and  some  protozoa  obtain  their  food 
by  diffusion  through  the  cell-membrane;  their  food-substances  must 
be  soluble  and  dissolved.  No  other  liquid  can  take  the  place  of  water. 

The  amount  of  water  required  by  microorganisms  cannot  be  stated 
briefly.  Several  factors  have  to  be  taken  into  consideration,  as  the 
osmotic  pressure,  the  insoluble  and  the  colloidal  substances,  the  species 
of  organisms,  temperature,  and  perhaps  others.  (See  pp.  184,  203.) 

AMOUNT  OF  FOOD  REQUIRED 

The  amount  of  food  that  is  ordinarily  decomposed  by  microorgan- 
isms and  the  amount  that  is  absolutely  necessary,  differ  widely.  The 
quantity  of  organic  and  inorganic  matter  just  sufficient  to  support  a 
very  weak  growth  is  certainly  very  small,  since  a  few  species  will 
multiply  to  some  extent  in  ordinary  distilled  water.  Such  water,  after 
having  stood  for  some  time,  is  found  to  contain  several  thousand 
bacteria  per  c.c.  It  may  seem  to  the  layman  that  in  such  water  it 
would  be  possible  to  detect  easily  the  organic  and  inorganic  matter  of 
the  microorganisms  so  that  it  could  not  be  considered  distilled  water. 
An  estimate  of  the  weight  of  bacteria  demonstrates,  however,  that  this 
is  not  the  case.  If  we  suppose  the  average  bacterial  cell  to  be  a 
cylinder  whose  base  measures  i  square  micron  and  whose  height  is  2 
microns  (which  is  a  high  estimate)  the  volume  of  such  a  cell  would  be 
1X1X2  cubic  microns  =  o.ooi  X  o.ooi  X  0.002  mm.  =  o.ooo,- 

*  Prepared  by  Otto  Rahn. 

221 


222  NUTRITION   AND    METABOLISM 

000,002  cu.  mm.  The  specific  gravity  of  bacteria  being  very  nearly  i, 
the  weight  of  one  bacterium  would  be  0.000,000,002  mg.;  100,000  cells 
per  c.c.  means  100,000,000  cells  per  liter,  which  would  weigh  0.2  mg. 
Of  this  total  weight,  at  least  four-fifths  is  water  and  only  one-fifth  is 
solid  matter.  The  total  solid  matter  in  i  liter  of  water  containing 
100,000  bacteria  per  c.c.  amounts  to  the  immeasurable  quantity  of 
0.04  mg.  Such  water  will  pass  the  tests  for  distilled  water.  How 
much  food  the  bacteria  in  distilled  water  have  used  is  impossible  to  say, 
since  besides  the  traces  of  minerals  in  the  water,  they  obtain  some  food 
from  volatile  compounds  of  the  air  like  carbon  monoxide  (CO), 
carbon  dioxide  (CO2),  ammonia  (NH3),  hydrogen  (H),  and  perhaps 
methane  (CH4).  Under  all  circumstances  the  amount  of  food  used  is 
very  small. 

On  the  other  extreme,  the  maximum  amount  of  food  cannot  be 
stated  very  definitely.  Usually  bacteria  cease  to  cause  decomposition 
because  of  the  accumulation  of  noxious  metabolic  products.  The 
ordinary  bacterium  from  sour  milk  will  not  form  more  than  about  one 
per  cent  of  lactic  acid,  because  this  is  the  highest  acid  concentration 
that  this  bacterium  can  endure.  If  this  acid  is  neutralized,  the  in- 
hibiting cause  is  removed,  and  the  lactic  fermentation  starts  anew 
until  the  maximum  acidity  is  reached  again.  The  amount  of  food 
decomposed  depends  largely  upon  the  power  of  the  organism  to  resist 
its  own  products.  If  the  food  is  too  concentrated,  however,  physical 
influences  may  interfere  with  the  metabolism  of  the  cell  (page  254). 

FOOD  FOR  GROWTH 

The  total  weight  of  a  large  bacterial  cell  is  estimated  in  the  pre- 
ceding paragraph  to  be  about  0.000,000,002  mg.,  of  which  only  about 
one-fifth  is  dry  matter.  The  smallest  quantity  that  can  be  weighed 
accurately  on  ordinary  analytical  balances  is  o.i  mg.  This  corre- 
sponds to  about  250,000,000  bacteria.  MacNeal  and  associates  found 
that  the  dry  matter  of  550,000,000  cells  of  B.  coli  weigh  o.i  mg.  The 
amount  -of  food  that  is  used  as  the  building  material  for  the  cell  is 
probably  larger  than  the  weight  of  the  cell  itself,  since  there  will  always 
be  present  waste  products,  but  it  is  of  the  same  order  of  magnitude,  i.e., 
very  small  and  often  hardly  measurable.  The  example  of  the  urea  fer- 
mentation (page  202)  illustrates  this  point  very  well. 

SOURCES  OF  CARBON. — The  compounds  which  can  serve  as  building 
stones  for  the  cell  vary  greatly  with  the  species.  The  source  of  carbon 


FOOD    OF   MICROORGANISMS  223 

for  all  green  plants  is  carbon  dioxide  (COa).  Animals  cannot  use  this, 
for  they  all  require  complex  compounds,  such  as  carbohydrates,  fats 
or  amino-acids.  Bacteria  exist  between  the  plants  and  animals  in 
this  respect.  Some  bacteria  have  already  been  mentioned  (page  201) 
as  being  able  to  use  carbon  dioxide  (CO2),  as  the  only  source  of  carbon; 
they  are  the  mineral-oxidizing  species.  Such  bacteria  are  called 
autotrophic  in  their  relation  to  carbon,  since  they  use  it  in  the  inorganic 
form.  A  bacterium  feeding  on  carbon,  as  such,  would  be  called 
prototrophic;  bacteria  of  this  class  are  said  to  exist.  The  vast  majority 
of  microorganisms  are  heterotrophic,  using  carbon  in  organic  form, 
Organic  acids  and  sugars  are  excellent  sources  of  carbon  for  micro- 
organisms, although  proteins  and  their  decomposition  products  seem 
to  be  equally  satisfactory  as  construction  material. 

SOURCES  OF  NITROGEN. — The  sources  of  nitrogen  are  equally  varied; 
the  green  plants  use  nitrates;  animals  must  have  a  number  of  different 
amino-acids;  the  microorganisms  again  are  found  between  plants  and 
animals.  We  know  autotrophic  bacteria,  and  especially  molds  and 
yeasts  which  can  grow  with  nitrates  or  ammonium  salts  as  the  only 
source  of  nitrogen.  There  are  three  groups  of  prototrophic  bacteria 
in  their  relation  to  nitrogen — the  B.  amylobacter  group,  the  Ps.  radicicola 
group  and  the  Azotobacter  group.  These  bacteria  are  of  the  greatest 
importance  to  agriculture;  soil  fertility  depends,  to  a  large  extent, 
upon  the  last  two  groups,  for  they  take  nitrogen  gas  from  the  surround- 
ing air,  form  their  own  protoplasm  from  it,  and  thus  increase  the 
amount  of  chemically  combined  nitrogen  in  the  soil.  Details  of  their 
relation  to  soil  fertility  can  be  found  in  Chap.  Ill,  page  400.  The 
majority  of  bacteria  are  heterotrophic,  requiring  organic  nitrogen.  Urea 
is  not  well  adapted  for  this  purpose;  amino-acids  or  the  peptones  from 
which  amino-acids  are  derived  are  the  best  compounds  for  most 
organisms.  Asparagin  is  very  commonly  used  if  for  some  reason 
peptones  are  to  be  omitted. 

SOURCES  OF  HYDROGEN  AND  OXYGEN. — The  sources  of  hydrogen  are 
hardly  ever  discussed  with  bacteria  since  hydrogen  bears  such  a  close 
and  peculiar  relation  in  water  and  organic  food  supplies.  The  ulti- 
mate association  of  hydrogen  with  oxygen  in  the  molecule  of  water 
(H2O)  and  with  carbon  in  organic  substances  (CH4)  establishes  its 
importance  in  all  life  processes.  There  are  many  prototrophic  bacteria, 
using  oxygen  as  such;  others  are  able  to  reduce  such  compounds  as 


224  .  NUTRITION   AND    METABOLISM 

nitrates  or  sulphates,  which  would  be  autotrophic,  thus  providing  for 
their  needs.  Heterotrophic  bacteria  are  not  unusual.  In  this  connec- 
tion it  may  be  said  that  it  is  often  difficult  to  distinguish  between  oxy- 
gen needed  for  cell  construction  and  oxygen  needed  for  energy  formation. 
SOURCES  OF  MINERALS. — The  amount  of  mineral  matter  necessary 
for  the  construction  of  the  cell  is  very  small;  potassium  and  phos- 
phorus seem  to  be  among  the  most  essential  elements.  It  is  customary 
to  consider  a  tap  water  with  0.02  per  cent  of  di-potassium  hydro- 
gen phosphate  (K2HPO4),  sufficient  in  mineral  matter  of  all  kinds  to 
provide  for  fair  growth.  Some  of  the  common  materials  used  in  the 
preparation  of  nutrient  media,  such  as  meat  extract  and  peptone,  also 
contain  considerable  amounts  of  mineral  matter. 

FOOD  FOR  ENERGY 

As  all  food  in  its  decomposition  results  in  products  of  some  form  or 
other,  it  may  not  seem  justifiable  to  separate  a  paragraph  on  food 
from  another  on  products.  The  essential  difference  lies  in  the  fact  that 
we  consider  food  from  the  viewpoint  of  the  cell,  while  products  are 
commonly  considered  apart  from  the  construction  processes  of  the  cell 
and  only  from  their  application,  or,  it  may  be,  from  the  viewpoint  of 
usefulness  to  man. 

Animals  provide  for  their  energy  by  oxidations,  and  almost  exclu- 
sively by  complete  oxidations.  Some  bacteria,  and  most  molds,  do 
the  same.  The  range  of  materials  which  can  serve  as  food  for  this  pur- 
pose is  surprising.  With  animals,  the  food  is  practically  limited  to 
plant  and  animal  tissue.  With  bacteria,  we  find  the  strangest  sub- 
stances, such  as  hydrogen,  carbon  monoxide,  coal,  marsh  gas,  hydrogen 
sulphide,  ammonia,  nitrites,  formic  and  oxalic  acids,  alcohol  and  thio- 
sulphates  serving  this  purpose.  The  fact  that  many  gases  are  used 
as  food  makes  us  realize  that  oxygen  is  not  such  an  extraordinary 
compound  as  animal  physiology  seems  to  indicate,  but  that  it  should  be 
classed  merely  as  one  of  the  many  food  compounds.  This  is  especially 
significant  since  it  will  be  shown  later  that  free  oxygen  is  not  necessary 
for  microbial  life,  and  that  many  organisms  can  exist  without  it. 

The  oxidations  are  not  always  complete.  The  formation  of  nitrous 
acid  from  ammonia,  the  oxidation  of  alcohol  to  acetic  acid  are  such 
examples.  Some  organisms  are  highly  specialized  in  their  food  require- 
ments, especially  the  mineral-attacking  bacteria  are  usually  limited 
to  one  source  of  energy.  The  microorganisms  oxidizing  organic  com- 


FOOD    OF   MICROORGANISMS  225 

pounds  have,  as  a  rule,  the  ability  to  decompose  several  compounds, 
and  some  bacteria  are  common  scavengers,  able  to  feed  on  organic  acids, 
sugars,  fats  and  proteins. 

Oxygen  Relations. — It  is  characteristic  of  many  microorganisms  to 
provide  for  their  energy  without  using  free  oxygen.  One  such  example 
has  already  been  given  in  urea  fermentation. 

(NH2)2  CO  +  2H20  =  (NH4)2C03 

Urea  Ammonium  carbonate 

Very  common  is  the  decomposition  of  sugars  without  oxygen. 
The  two  most  typical  fermentations  of  this  type  are  the  alcoholic  and 
the  lactic  fermentations. 

C6H12O6  =  2C2H5OH  +  2CO2  +  22  Cal. 

Sugar  Alcohol 

C6H12O6  =  2C3H6O3  +  15  Cal. 

Sugar  Lactic  acid 

In  fermentations  of  this  type,  the  changes  take  place  without  an 
oxygen  gas  partaking  in  the  reactions.  These  fermentations  seem  to 
be  essentially  reactions  of  the  oxygen  atoms  within  the  sugar  molecule. 
One  side  of  the  molecule  is  reduced  while  the  other  side  is  oxidized. 
In  the  sugar  molecule,  each  carbon  atom  has  one  oxygen  atom.  In 
the  products  of  fermentation,  carbon  dioxide  has  two  oxygen  atoms  to 
one  carbon  atom,  and  in  alcohol  there  is  only  one  oxygen  atom  for  two 
carbon  atoms.  In  the  lactic  fermentation,  the  oxygen,  which  is  dis- 
tributed evenly  in  the  sugar,  is  shifted  to  one  side  of  the  molecule  in 
lactic  acid. 

H    H    H    H    H    O 
O    O    O    O    O     || 
Dextrose,        H— C— C— C— C— C— C 
H    H    H    H    H    H 
H        H       O 
O       || 

Alcohol,  HC— CH       C        Carbon  dioxide, 

H       H        || 
O 

H    H 
H    O    O 

Lactic  acid,  HC— C— C 

H    H     || 

O 

15 


226  NUTRITION   AND    METABOLISM 

In  some  of  the  more  complex  fermentations,  we  find  simultaneous 
formation  of  hydrogen  or  methane  and  carbon  dioxide;  the  one  is 
the  end  product  of  reduction,  the  other  the  product  of  complete  oxida- 
tion. This  also  indicates  that  the  oxidation  of  one  part  of  the  molecule 
takes  place  at  the  expense  of  the  other. 

In  a  similar  way,  some  organic  acids,  e.g.,  tartaric  and  lactic  acids, 
can  be  fermented  by  certain  bacteria  without  requiring  oxygen.  Some 
bacteria  have  the  ability  to  attack  proteins  and  decompose  them 
completely  in  the  absence  of  oxygen. 

Bacteria,  having  the  ability  to  provide  for  their  energy  without 
oxygen  gas,  may  live  in  the  complete  absence  of  oxygen,  and  may 
multiply  indefinitely  without  it  as  long  as  there  is  sufficient  food.  But 
some  microorganisms,  such  as  yeasts,  seem  to  grow  only  for  a  limited 
time  in  the  absence  of  oxygen.  Finally,  they  cease  growing,  and 
we  may  well  assume  that  they  need  oxygen  for  cell  construction  which 
can  be  used  in  no  other  form  except  as  molecular  oxygen.  The  urea 
bacteria  also  belong  in  this  group. 

A  large  number  of  bacteria  and  yeasts,  and  also  a  few  molds,  can 
provide  for  their  energy  by  either  oxidation  or  decomposition  in  the 
absence  of  oxygen.  Very  commonly  a  great  variety  of  compounds  can 
be  found  which  may  be  oxidized  while  but  very  few  can  be  intra- 
molecularly  fermented  without  oxygen.  This  is  easily  understood: 
all  organic  compounds  will  yield  heat  upon  oxidation,  while  exothermic 
intramolecular  changes  require  a  special  structure.  Carbohydrates 
are  the  most  excellent  substances  for  such  intramolecular  decomposi- 
tions. S.  cerevisice  and  B.  coll  can  live  in  sugar-free  broth  only  if  ex- 
posed to  the  air.  They  provide  for  all  their  needs  by  oxidation  of  the 
protein.  If  oxygen  is  excluded,  growth  depends  upon  sugar,  or  a 
similar  fermentable  compound.  We  test  for  the  absence  of  sugar  in  a 
given  solution  by  pouring  it  in  a  fermentation  tube  and  inoculating 
with  B.  coll:  if  the  liquid  in  the  closed  arm  remains  clear,  i.e.,  if  B.  coll 
does  not  grow  without  oxygen,  it  is  a  good  indication  that  no  sugar  is 
present. 

It  is  usually  assumed  that  in  fermentations  of  this  nature,  the 
oxygen  atoms  are  shifted  within  the  same  molecule.  In  other  cases, 
oxygen  is  taken  from  one  molecule  and  used  for  the  oxidation  of 
another.  This  results  in  one  of  the  molecules  being  reduced.  Nitrates 
are  reduced  in  this  way  to  nitrites,  or  ammonia,  or  nitrogen  gas;  sul- 


FOOD   OF  MICROORGANISMS  227 

/ 

phates  to  hydrogen  sulphide,  and  litmus  or  methylene  blue  to  the 
colorless  leuco-compounds.  Such  removal  of  oxygen  from  a  molecule 
requires  energy,  and  is  possible  only  when  the  bacterium  by  using  the 
oxygen  for  oxidation  of  organic  matter  can  obtain  a  larger  amount 
of  energy.  The  following  example  shows  such  a  possibility: 

2KNO3  +  36.6  Cal.  =  2KNO2  +  O2 
C2H5OH  +  Oa  =  CH3CO2H  +  H2O  +  115  Cal. 

This  process  leaves  an  energy  balance  of  115  —  36.6  =  78.4  Cal.  for 
the  needs  of  the  bacterium. 

Such  decompositions  are  sometimes  referred  to  as  "reducing  fermen- 
tations" but  this  term  is  not  correct,  as  the  reduction  must  always  be 
accompanied  by  a  simultaneous  oxidation  process. 

The  amount  of  energy  liberated  by  a  fermentation  without  oxygen 
is  much  smaller  than  that  furnished  by  complete  oxidation;  the  intra- 
molecular change  always  leaves  organic  compounds  which  contain  a 
considerable  amount  of  the  total  energy.  Yeast,  in  presence  of  very 
much  oxygen,  oxidizes  sugar  completely  to  water  and  carbon  dioxide. 

C6H12O6  +  120  =  6CO2  +  6H2O  +  674  Cal. 

while  in  the  absence  of  oxygen  it  will  change  the  sugar  to  alcohol  and 
carbon  dioxide. 

C6H12O6  =  2C2H5OH  +  2CO2  +  22  Cal. 

The  energy  gained  in  the  first  process  is  about  thirty  times  as  large 
as  that  gained  in  the  second  process.  This  was  demonstrated  as  early 
as  1861  by  Pasteur.  He  grew  yeast  in  sugar  solutions,  varying  only 
the  amount  of  oxygen  in  contact  with  the  medium.  At  the  end  of 
the  experiment,  the  weight  of  the  dry  yeast  and  the  decomposed  sugar 
was  determined,  and  the  amount  of  sugar  necessary  to  produce  one 
part  of  yeast  was  computed.  He  found: 

In  a  closed  flask,  without  any  air i  part  yeast  required  1 76  parts  sugar. 

In  a  closed  flask,  with  large  air  space i  part  yeast  required    23  parts  sugar. 

In  a  thin  layer,  a  few  mm.  thick i  part  yeast  required      8  parts  sugar. 

In  a  very  thin  layer,  in  24  hours i  part  yeast  required      4  parts  sugar. 

This  experience  led  Pasteur  to  the  conclusion  that  fermentation 
corresponded  to  the  respiration  process  of  animals,  that  fermentation 
was  respiration  without  oxygen. 

It  is  quite  evident  that  since  the  utilization  of  the  food  in  the 


228  NUTRITION   AND    METABOLISM 

absence  of  oxygen  is  very  high,  the  organisms  have  to  decompose 
much  more  food.  This  accounts,  to  a  great  extent,  for  the  enormous 
destructive  power  of  bacteria,  when  comparisons  of  the  great  quantity 
of  food  decomposed  are  made  with  the  very  insignificant  weights  of 
cells.  It  has  been  estimated  that  the  lactic  bacteria  decompose  their 
own  weight  of  sugar  in  one  hour. 

Summing  up  the  relation  of  oxygen  to  microorganisms,  some 
bacteria,  and  especially  the  molds,  are  found  depending  upon  oxygen  as 
an  indispensable  part  of  their  food.  Three  groups  are  recognized: 
Those,  a  large  number,  organisms  in  the  presence  of  oxygen  producing 
oxidations;  those  able  to  sustain  life  without  oxygen;  and  those  de- 
pending entirely  upon  decompositions  which  require  no  oxygen. 
The  lactic  bacteria  and  the  butyric  bacteria  belong  in  the  last  group. 

In  considering  the  oxygen  requirements,  it  is  customary  to  in- 
clude another  influence  of  oxygen  upon  bacteria.  This  has  really 
nothing  to  do  with  its  food  value,  but  deals  with  the  poisonous  qualities 
of  oxygen.  Oxygen  in  this  light  may  well  be  called  a  poison  as  it  will 
kill  bacteria  in  very  low  concentrations.  Ordinarily  it  is  regarded  as 
constituting  over  20  per  cent  of  our  atmosphere.  But  if  a  study  is 
made  of  its  effect  upon  bacteria,  it  is  necessary  to  measure  it  in  the 
same  way  food  is  measured,  and  consider  the  concentration  in  which 
it  is  offered  to  the  cell.  Microorganisms  obtain  their  oxygen  not  as 
gas,  but  as  dissolved  oxygen.  The  solubility  of  oxygen  is  very  small, 
about  0.0009  Per  cent  at  20°.  Practically  all  bacteria  die  readily  if  the 
oxygen  concentration  is  raised  to  thirty  times  the  atmospheric  pressure. 
This  would  mean  a  concentration  of  0.027  per  cent.  It  shows  that 
oxygen  is  about  as  poisonous  as  formaldehyde  or  bichloride  of  mercury. 

Some  bacteria  are  extremely  sensitive  to  oxygen,  and  will  die  if 
exposed  to  ordinary  atmospheric  oxygen.  They  grow  only  if  oxygen 
is  almost  completely  removed.  These  organisms  are  called  the 
strictly  anaerobic  or  obligate  anaerobic  bacteria.  They  are  contrasted 
with  the  facultative  anaerobic  bacteria  which  thrive  with  oxygen  as  well 
as  without,  and  the  strictly  aerobic  bacteria  which  have  to  have  oxygen 
for  their  normal  life  processes. 

No  strict  limits  can  be  drawn  between  aerobic  and  anaerobic 
bacteria.  Even  the  most  sensitive  of  organisms  will  be  able  to  tolerate 
traces  of  oxygen,  while  the  strictly  aerobic  bacteria  can  multiply  also 
if  the  oxygen  concentration  is  below  that  of  a  saturated  solution.  The 


FOOD    OF    MICROORGANISMS 


22Q 


limits  of  growth  for  the  anaerobic  bacteria  are  the  limits  of  tolerance  of 
the  poisoning  oxygen;  the  lower  limit  of  growth  for  the  aerobic  bacteria 
is  a  question  of  too  scanty  food  supply.  The  relation  between  bacteria 
and  oxygen  is  graphically  represented  in  the  following  diagram,  after 
Kruse: 


Oxygen  Pressure* 
0       0."L     O.V      O.k     O.t      I.O 


2.0 


3.0 


FIG.  112. — Influence  of  oxygen  upon  microorganisms. 

The  lines  indicate  the  oxygen  concentrations  where  growth  is  possible.  Line 
i  is  a  strict  anaerobe;  2  is  not  quite  so  strict;  3  is  still  less  sensitive  though  it 
cannot  grow  if  exposed  to  direct  influence  of  the  atmosphere;  4  is  a  facultative 
bacterium  such  as  B.  coli;  5  is  another  one  which  can  tolerate  still  more  oxygen; 
6  can  grow  only  with  oxygen  but  can  get  along  with  very  little:  it  might  be  one 
of  the  urea  bacteria;  8  is  more  dependent  upon  oxygen  and  the  line  would  corre- 
spond to  average  molds;  7  is  a  peculiar  type  needing  oxygen  and  yet  being  very 
sensitive  to  it.  The  sulphur  bacteria,  e.g.,  the  Beggiatoacecs,  belong  to  type  7.  Type 
9  is  said  to  be  representative  of  B.  abortus. 


1.0  indicates  the  normal  atmospheric  oxygen  content  (about  21  per  cent  by  volume). 


CHAPTER  IV 

PRODUCTS  OF  MICROBIAL  ACTIVITIES* 
GENERAL  CONSIDERATIONS 

The  great  difference  in  the  metabolism  of  animals  and  of  bacteria, 
even  though  they  feed  essentially  on  the  same  foods,  is  the  incomplete 
metabolism  of  most  bacteria,  contrasting  sharply  against  the  very 
complete  oxidation  of  food  in  the  animal  body.  The  food  of  the  animal 
is  decomposed  by  the  body  cells  to  carbon  dioxide,  water  and  urea.  It 
is  the  most  complete  decomposition  possible,  excepting  urea  which, 
however,  is  very  near  the  final  decomposition  product,  ammonium 
carbonate.  Microorganisms,  on  the  contrary,  are  characterized  by 
incomplete  metabolism.  They  do  not  commonly  oxidize  their  food  to 
the  end  products  but  many  of  them  produce  organic  compounds  which 
are  not  farther  decomposed  by  them.  It  is  this  partial  decomposition 
of  organic  matter  which  makes  microorganisms  play  such  an  important 
role  in  life  and  industries.  Our  modern  microbiology  is  dated  from  the 
time  when  Pasteur  showed  that  the  alcohol  in  the  beer  fermentation, 
the  lactic  acid  in  the  souring  of  milk,  the  acetic  acid  in  the  vinegar 
fermentation  are  products  of  microbial  activity.  The  existence  of 
microorganisms  had  been  known  for  nearly  200  years,  but  they  were 
considered  largely  as  a  curiosity;  as  soon  as  they  were  recognized  as 
the  cause  of  fermentations,  and  of  toxins,  they  received  at  once  the 
greatest  attention.  Not  all  bacteria  cause  incomplete  decompositions ; 
some  oxidize  as  completely  as  animals  do.  Others,  again,  form  first 
intermediary  products,  which  they  later  decompose  completely;  among 
these,  are  found  many  molds,  the  sulphur  bacteria,  and  some  species  of 
the  vinegar  bacteria. 

THE  CHEMICAL  EQUATIONS  OF  FERMENTATIONS 

The  metabolism  of  all  organisms  is  considered  to  be  a  chemical 
process  which  follows  in  most  respects  the  laws  of  chemistry.  That 
we  are  not  familiar  with  all  the  changes  taking  place  in  the  cell  is  not 

*  Prepared  by  Otto  Rahn. 

230 


PRODUCTS   OF   MICROBIAL  ACTIVITIES  231 

because  we  are  dealing  with  unknown  forces,  but  simply  because  we 
do  not  know  all  the  factors  involved  in  the  process.  Some  of  the 
chemical  changes  caused  by  the  living  cell  can  be  imitated  exactly  by 
the  chemist  in  a  test-tube.  This  may  be  illustrated  by  the  oxidation 
of  alcohol  to  acetic  acid,  the  decomposition  of  urea  to  ammonium 
carbonate  and  of  ammonia  to  nitrate.  Some  other  processes  are  not 
as  fully  understood  and  not  as  easily  imitated.  The  alcoholic  and 
acid  fermentations  of  sugars  are  of  such  nature.  There  is  no  reason 
to  suppose,  however,  that  these  processes  are  other  than  chemical 
changes.  Since  a  chemical  process  can  always  be  expressed  by  a 
chemical  equation,  we  should  expect  the  same  with  the  fermentations 
and  decompositions  caused  by  microorganisms. 

This  formulation  is  not  always  simple,  because  the  greater  number 
of  microorganisms  decompose  organic  substances  in  more  than  one  way. 
Also,  certain  compounds  may  be  produced  in  such  small  quantities  as 
to  escape  the  chemical  analysis  entirely,  since  the  determination  of 
many  organic  compounds  is  a  very  difficult  task.  Again,  part  of  the 
decomposed  material  will  usually  be  assimilated  in  the  growth  of  the 
cells;  hence  more  material  disappears  than  can  be  accounted  for  by 
the  fermentation  products.  There  are  several  possibilities  for  dis- 
crepancies; accurate  equations  can  be  given  only  for  the  simplest  fer- 
mentations, the  products  of  which  can  be  analyzed  more  or  less  exactly. 

The  best  studied  microbial  process  is  the  alcoholic  fermentation. 
The  simplest  equation  for  the  decomposition  of  dextrose  into  alcohol 
and  carbon  dioxide  by  yeast  is 

C6H1206  =  2C2H5OH  +  2CO2 

180  92  88 

According  to  this  formula,  100  parts  of  dextrose  should  give  51.11  parts 
of  alcohol  and  48.89  parts  of  carbon  dioxide.  The  actual  yield  comes 
very  close  to  these  numbers,  but  does  not  reach  them;  the  largest 
amounts  found  were  46-47.5  per  cent  of  carbon  dioxide  and  47.5-48.67 
per  cent  of  alcohol.  Under  the  most  favorable  conditions,  the  total 
yield  of  the  products  of  fermentation  was  only  95  per  cent  of  the 
theoretical  yield. 

Other  products  are  formed  besides  the  alcohol  and  carbon  dioxide. 
The  amount  of  glycerin  found  in  fermented  liquids  varies  very  much 
with  the  conditions  of  fermentation;  it  reaches  from  1.6  to  13.8  per  cent 


232  NUTRITION   AND    METABOLISM 

of  the  alcohol  or  from  0.8  to  6.9  per  cent  of  the  fermented  sugar.  A 
small  quantity  of  succinic  acid  is  also  formed,  usually  about  0.6  to  0.7 
per  cent  of  the  fermented  sugar.  Traces  of  acetic  acid  and  of  lactic 
acid  seem  to  be  normal  products  of  the  process  of  fermentation,  and  we 
always  find  fusel  oil.  The  latest  investigations  seem  to  indicate  that 
glycerin  and  succinic  acid  are  produced  by  yeast  cells  even  in  the  absence 
of  sugar.  This  discovery  makes  it  probable  that  the  glycerin  and  suc- 
cinic acid  are  derived  from  the  reserve  substances  of  the  yeast  cells, 
such  as  lecithin,  and  are  not  direct  products  of  fermentation.  This 
accounts  also  for  the  variation  of  the  proportion  between  alcohol  and 
glycerin.  Fusel  oil  is  now  believed  to  be  a  waste  product  of  cell 
construction. 

Similar  are  the  experiences  with  the  lactic  fermentation  which  has 
been  studied  almost  as  extensively  as  alcoholic  fermentation.  If  it  is 
supposed  that  the  formation  of  lactic  acid  follows  the  equation 

Ci2H22Oii  +  H2O  =  4C3H6O3 

342  18  360 

Lactose  Lactic  acid 

the  actual  yield  of  acid  is  found  to  be  between  90  per  cent  and  98  per 
cent  of  the  theoretical.  The  other  2-10  per  cent  are  either  used  for 
cell-growth  or  for  products  which  thus  far  have  escaped  chemical  de- 
termination. Small  discrepancies  will  also  be  found  in  fermentation 
of  urea  and  in  the  nitrifying  process,  where  small  amounts  of  the 
nitrogenous  material  are  used  for  cell-growth. 

Another  difficulty  in  finding  the  chemical  equation  of  a  microbial 
fermentation  is  the  fact  that  this  process  may  change  with  the  age  of  the 
culture.  In  those  fermentations  where  several  gases,  as  carbon  dioxide 
and  hydrogen,  are  produced,  the  relative  proportion  of  the  two  is  not 
always  constant.  In  the  butyric  fermentation  of  dextrose  by  B. 
amylozyma,  Perdrix  tries  to  account  for  this  change  by  assuming  three 
different  phases  of  the  process  at  various  ages  of  the  cultures,  repre- 
sented by  the  following  equations: 
First  stage:  56C6Hi2O6  +  42H2O  =  ii6H2  +  ii4CO2  +  3oCH3COOH  + 

Dextrose  Acetic  acid 

36CH3CH2CH2COOH. 

Butyric  acid 

Second  stage:  46C6Hi2O6  +  i8H2O  =  ii2H2  +  94CO2  +  i5CH3COOH  + 

38CH3CH2CH2COOH. 
Third  stage:  C6Hi2O6  =  2H2  +  2CO2  +  CH3CH2CH2COOH. 


PRODUCTS    OF    MICROBIAL   ACTIVITIES  233 

Kruse  has  called  attention  to  the  fact  that  these  complex  equations 
can  well  be  explained  as  the  simultaneous  occurrence  of  the  following 
simple  fermentations: 

C6H12O6  =  2H2  +  2CO2  +  CH3CH2CH2CO2H 

C6H12Oft  =  3CH3CO2H 

C6Hi2O6  -f-  6H2O  =  6CO2  +  i2H2 

The  first  fermentation  continues  when  the  others  have  already  ceased, 
and  thus  the  last  stage  of  Perdrix's  equations  is  very  simple.  Brede- 
mann  also  found  that  the  proportion  of  the  various  products  formed  by 
B.  amylobacter  varies  greatly  with  the  conditions,  and  the  same  has  been 
recently  established  in  the  fermentation  of  B.  coli. 

Other  complications  occur  when  an  organism  is  able  to  use  its  own 
products  as  food,  as  is  the  case  with  some  acetic  bacteria.  They  will 
at  first  produce  considerable  amounts  of  acetic  acid  and  after  a  while 
they  oxidize  the  acid  completely.  It  becomes  impossible  to  account  for 
microbial  activity  by  a  chemical  equation  when  several  organic  com- 
pounds are  decomposed  at  the  same  time  as  is  found  to  occur  in  some 
foods,  as  butter,  cheese,  ensilage  and  in  sewage.  It  is  also  impossible 
to  formulate  exactly  decompositions  which  are  caused  by  mixed  cultures. 
The  complications  become  so  great  and  the  relations  between  different 
organisms  are  so  little  known  that  it  is  useless  to  make  the  attempt. 

PRODUCTS  FROM  NITROGEN-FREE  COMPOUNDS 

SUGARS. — It  would  be  entirely  beyond  the  limits  of  this  book  to 
give  an  account  of  all  the  different  ways  in  which  sugars  and  other 
compounds  can  be  decomposed  by  microorganisms.  It  is  much  more 
important  for  the  beginning  microbiologist  to  acquaint  himself  with 
the  main  types  of  sugar  fermentations  and  with  the  characteristics 
of  the  organisms  which  bring  about  these  changes. 

In  the  action  of  microorganisms  many  distinguish  somewhat  crudely 
six  common  types: 

Complete  oxidation. 

Partial  oxidation. 

Alcoholic  fermentation. 

Lactic  fermentation. 

Acid  gas  fermentation. 

Butyric  fermentation. 
Most  of  these  types  have  been  mentioned  previously. 


234  NUTRITION  AND   METABOLISM 

Complete  oxidation  of  carbohydrates  is  observed  most  commonly 
among  molds  and  mycodermas,  and  also  in  a  few  bacteria,  e.g.,  in  Azoto- 
bacter.  It  is  possible  only  where  there  is  a  ready  oxygen  supply,  as, 
e.g.,  in  soils  of  an  open  texture,  in  trickling  niters,  and  on  the  surface 
of  decaying  fruits. 

The  incomplete  oxidation  is,  as  a  rule,  more  common  in  nature. 
Frequently  microorganisms  produce  first  an  incomplete  oxidation,  but 
later  oxidize  the  intermediate  products  completely.  The  molds  are 
typical  examples.  Aspergillus  niger  is  noted  for  its  formation  of  oxalic 
acid.  If  it  is  grown  in  a  sugar  solution,  it  will  bring  about  at  first  a 
rapid  increase  in  acidity,  but  after  a  while,  it  decreases  again,  when  the 
acid  is  oxidizing  completely.  The  following  processes  may  be  noted: 

C6H1206  +  90  =  3(C02H)2  +  3H20 

Oxalic  acid 

(C02H)2  +  O  =  2  C02  +  H20 

The  intermediate  product  can  be  accumulated  by  precipitating  it  with 
lime  which  neutralizes  the  acidity.  This  principle  is  used  in  the  com- 
mercial manufacture  of  citric  acid  by  Citromyces,  a  mold  closely 
related  to  the  genus  Penicillium.  This  mold  oxidizes  sugar  to  citric 
acid  according  to  the  following  equation: 


C6H1206  '+  30  =  CeHsOy  +  2H20 

Citric  acid 

This  fermentation  is  much  more  complicated  than  this  equation  indi- 
cates, on  account  of  the  entirely  different  chemical  structures  of  citric 
acid  and  dextrose.  The  practical  yield  in  the  factory  is  only  about  one- 
half  of  the  theoretical,  since  complete  oxidation  cannot  be  avoided 
altogether. 

The  oxidation  processes,  just  recited,  can  take  place  only  in  the 
presence  of  oxygen;  the  other  four  types  of  carbohydrate  decomposi- 
tion require  no  oxygen,  and  take  place  as  well  in  the  absence  of  oxygen; 
the  butyric  fermentation  is  brought  about  only  in  the  absence  of  oxygen. 

Alcoholic  fermentation  is  caused  only  by  yeasts  and  a  few  molds; 
no  bacterium  produces  alcohol  according  to  the  well-known  equation 
mentioned  above.  Alcohol  is  formed  by  several  bacteria  but  only  in 
small  quantities  and  always  together  with  several  acids;  this  is  a 
distinctly  different  type  of  decomposition. 

In  the  above  groups  and  the  following  groups  of  microorganisms, 
there  appears  to  be  a  close  agreement  between  the  morphological 


PRODUCTS    OF   MICROBIAL   ACTIVITIES  235 

characters  of  the  organisms  involved  and  the  specific  type  of  fermenta- 
tion. Practically  all  the  alcoholic  organisms  are  yeasts,  and  the  lactic 
acid-producing  organisms  are  streptococci  or  closely  related  bacteria. 
The  lactic  bacteria,  as  they  are  briefly  named,  such  as  are  responsible 
for  lactic  fermentation,  are  readily  recognized  by  their  scanty  growth 
on  agar,  and  their  excellent  growth  in  milk,  bringing  about  a  solid 
curdling  in  one  to  three  days.  They  change  sugar  to  lactic  acid  only. 

C6H]206  =  2C3H6O3 

No  gas  and  no  volatile  acids  are  formed  by  these  bacteria.  The  best- 
known  representative  of  this  group  is  the  organism  which  causes  the 
normal  souring  of  milk.  It  was  originally  called  Bacterium  lactis  acidi, 
but  on  account  of  its  very  close  relation  to  the  streptococci,  it  is  more 
commonly  now  named  Streptococcus  lacticus.  Many  streptococci  will 
produce  the  true  lactic  fermentation. 

The  last  two  groups  of  organisms,  alcoholic  and  lactic,  represent 
complex  fermentations.  There  are  several  products  formed,  and  as  has 
already  been  pointed  out  in  the  paragraph  on  the  equation  of  fermen- 
tations, the  entire  fermentation  cannot  be  described  accurately  by  one 
equation,  for  different  fermentations  operate  independently  and  simul- 
taneously in  the  same  cell.  Under  slightly  different  experimental 
conditions  the  one  or  other  of  these  simultaneous  fermentations  may  be 
favored,  accordingly  a  varying  proportion  of  the  products  is  formed. 

The  typical  representatives  of  the  acid-gas  forming  group  of  micro- 
organisms which  cause  acid-gas  fermentation  are  B.  coli,  and  its  near 
relative,  Bact.  aerogenes.  Many  of  the  gas-formers  in  nature  belong  in 
this  group;  the  bacteria  of  the  fermentations  of  pickles,  sauerkraut, 
salt-rising  bread,  the  gassy  fermentation  of  milk  are  some  of  the  many 
representatives.  They  are  distinct  rods,  with  good  surface  growth, 
and  do  not  liquefy  gelatin.  They  are  commonly  spoken  of  as  the  coli- 
aerogenes  group.  Some  of  them  have  peritrichate  flagella,  while 
others  are  not  motile. 

The  fermentation  of  dextrose  brought  about  by  these  organisms 
has  been  described  originally  by  Harden  in  the  equation: 

2C3H1206  +  H20  =  2C3H603  +  CH3C02H  +  C2H6OH  +  2CO2  +  2H2 

Dextrose  Lactic  acid  Acetic  acid  Alcohol 

Harden  himself  stated  later  that  this  equation  holds  only  for  one 
strain,  and  that  we  have  several  different  strains  distinguished  by  a 


236  NUTRITION    AND    METABOLISM 

proportion  of  products  quite  different  from  the  one  suggested  by  the 
equation.  Recently  Kamm  has  shown  that  a  good  mineral  food 
(probably  phosphates  are  the  essential  agent)  favors  a  formation  of 
gas  and  of  volatile  acids,  while  a  scant  supply  of  minerals  causes  the 
bacteria  to  produce  mainly  lactic  acid.  We  must  assume,  therefore, 
at  least  two  simultaneous  independent  fermentations: 

C6Hi2O6  =  2C3H6O3 
and 

C6H12O6  +  H2O  =  CH3CO2H  -f-  CH3CH2OH  +  2CO2  +  2H2 

The  first  equation  is  already  known  to  us;  it  is  the  true  lactic  fermenta- 
tion. The  second  equation  may  be  divided  still  further  into  several 
simpler  equations. 

B.  typhosus,  causing  typhoid  fever,  is  closely  related  to  B.  coli,  but 
does  not  form  gas.  It  forms,  however,  formic  acid,  HC02H,  which,  if 
decomposed,  would  give  H2  +  C02. 

The  last  type  of  sugar  fermentations  is  the  butyric  fermentation, 
in  which  butyric  acid  is  the  most  conspicuous,  but  not  the  only  fermen- 
tation product.  Acetic  acid,  hydrogen  and  carbon  dioxide,  and,  with 
some  organisms  at  least,  ethyl  and  butyl  alcohols  are  formed  along  with 
butyric  acid.  As  already  mentioned  in  the  paragraph  on  the  equation  of 
fermentation,  Kruse  believes  this  fermentation  to  consist  of  several 
simultaneous  fermentations,  of  which  the  most  interesting  at  this  stage 
is  the  one  showing  the  formation  of  butyric  acid. 

C6H1206  =  2H2  +  2C02  +  C4H802 

The  organisms  producing  butyric  acid  are  mostly  strictly  anaerobic 
spore  formers  with  a  tendency  to  form  spindle-shaped  cells;  they  stain 
bluish-black  with  iodine  and  Bredemann  gave  the  clostridium  group 
one  species  name,  B.  amylobacter,  as  he  found  no  distinct  and  char- 
acteristic differences  between  the  many  strains  which  he  studied. 
Many  members  of  this  group  have  the  ability  to  fix  nitrogen,  i.e. 
to  build  up  their  protoplasm  without  using  any  sources  of  nitrogen 
other  than  nitrogen  gas.  Most  of  the  so-called  "Clostridium"  species 
belong  in  this  group.  Butyric  acid  is  also  formed  by  B.  tetani  and  by 
B.  botulinus,  the  latter  of  which  causes  the  most  dangerous  kind  of  meat 
poisoning. 

Of  other  sugar  fermentations  may  be  mentioned  here  only  by  name, 


PRODUCTS    OF    MICROBIAL   ACTIVITIES  237 

the  slimy  fermentations,  as  manifested  in  ropy  milk  and  the  mannit 
fermentation.  The  latter  is  one  of  the  very  few  reduction  processes 
brought  about  by  bacteria,  and  one  which  causes  trouble  in  wine. 

What  has  been  stated  broadly  for  sugars  holds  to  some  extent  true 
also  for  the  alcohols  derived  from  sugars,  including  glycerin.  Many 
bacteria  fermenting  dextrose  can  also  ferment  mannit  and  glycerin 
with  a  slight  variation  of  the  products,  but  some  do  not  do  this. 

Among  disaccharides  there  is  a  great  variation  of  fermentation. 
Some  groups  ferment  lactose  readily  as  the  coli  organisms  and  Strept. 
lacticus,  while  among  yeasts,  fermentation  of  lactose  is  rare.  Practi- 
cally all  yeasts  ferment  saccharose,  however,  and  among  the  lactic 
bacteria  and  the  coli  group  many  strains  cannot  ferment  saccharose. 

STARCH. — Quite  different  is  the  fermentation  of  the  insoluble  carbo- 
hydrates of  which  we  can  mention  only  starch  and  cellulose.  Insoluble 
compounds  can  be  fermented  only  after  being  made  soluble  by  an 
enzyme,  the  amylase  (see  mechanism  of  metabolism).  Amylase  is 
produced  by  most  molds,  by  none  of  the  fermenting  yeasts,  by  a  few 
torulas,  and  perhaps  mycodermas,  and  by  a  great  many  of  the  bacteria. 
The  sugar  thus  produced  from  starch  is  decomposed  according  to  the 
main  types  mentioned  under  sugars.  The  lactic  bacteria  and  the  coli 
bacteria  do  not  attack  starch,  but  some  acid-gas  fermentations  of 
starchy  foods  do  take  place.  Butyric  fermentation  of  starch  is  com- 
mon. Alcoholic  fermentation  can  be  accomplished  only  by  some  of 
the  Mucors,  and  Aspergilli. 

CELLULOSE  is  decomposed  only  by  very  few  organisms;  these  must 
be  very  active  and  very  numerous,  to  judge  from  the  enormous  amounts 
of  cellulose  produced  and  destroyed  every  year  on  earth.  Molds  and 
higher  fungi  play  probably  the  main  role  in  its  decomposition;  the 
products  have  not  been  determined,  but  we  may  well  assume  a  complete 
oxidation,  since  no  intermediate  products  have  ever  been  mentioned. 
No  yeast  is  known  to  decompose  cellulose,  and  among  the  bacteria  we 
find  but  very  few  species.  Some  species  have  recently  been  isolated 
which  decompose  cellulose  in  the  presence  of  air;  the  products  have  not 
been  determined;  we  can,  however,  assume  a  partial  oxidation,  eventu- 
ally a  complete  oxidation.  Besides  the  aerobic  fermentation,  we  have 
two  types  of  anaerobic  fermentation  which  are  ordinarily  described  as 
the  hydrogen  fermentation  and  the  methane  fermentation.  In  these 
fermentations  the  gases  mentioned,  together  with  carbon  dioxide,  are 


238  NUTRITION  AND   METABOLISM 

liberated,  and  butyric  and  acetic  acids  are  formed  at  the  same  time. 
The  marsh  gas  of  the  marshes  originates  in  this  way. 

Summing  up  all  the  products  formed  from  carbohydrates,  we  find 
several  acids,  among  them  lactic  and  acetic  acids  most  commonly, 
and  ethyl  alcohol,  rarely  other  alcohols,  besides  carbon  dioxide, 
hydrogen  and  water.  The  variety  is  not  so  great,  but  with  these  few 
compounds,  a  number  of  different  combinations  are  possible,  and  the 
complication  of  the  study  of  such  fermentations  lies  mostly  in  the 
simultaneous  formation  of  several  of  the  compounds. 

ACIDS  AND  ALCOHOLS. — The  organic  acids  and  alcohols  can  be 
decomposed  further  by  bacteria  and  molds,  also  by  some  yeasts,  to 
simpler  compounds.  Ordinarily,  this  decomposition  consists  in  the 
complete  oxidation.  Thus,  Oidium  lactis  will  destroy  the  lactic  acid 
of  sour  milk  and  of  soft  cheeses  by  complete  combustion. 

C3H603  +  60  =  3C02  +  3H20 

By  the  same  process,  the  acidity  of  sauerkraut,  ensilage,  pickles  is 
reduced  by  mycoderma  species.  Another  Mycoderma  is  known  to 
destroy  acetic  acid  and  thus  spoil  vinegar  or  fruits  and  vegetables  kept 
in  vinegar;  the  yeast  grows  in  a  thin,  dry  white  scum  over  the  surface, 
and  oxidizes  the  acetic  acid. 

CH3CO2H  +  4O  =  2CO2  +  2H2O 

The  oxidation  of  alcohols  is  not  always  complete.  Especially  ethyl 
alcohol  is  usually  oxidized  first  to  acetic  acid;  this  is  the  common  vinegar 
fermentation.  Many  different  kinds  of  vinegar  bacteria  are  known, 
some  forming  gelatinous  masses  of  cell  membranes  called  mother-of- 
vinegar,  while  others  remain  as  separate  small  cells.  They  all  oxidize 
alcohol  first  to  acetic  acid. 

CH3CH2OH  +  2O  =  CH3CO2H  +  H2O 

But  most  of  them  will  oxidize  later  the  acetic  acid  completely  to  carbon 
dioxide,  after  the  alcohol  is  all  exhausted,  unless  the  oxygen  supply  is 
shut  off.  This  behavior  reminds  one  of  the  formation  and  destruction 
of  oxalic  acid  by  Aspergillus,  mentioned  previously.  It  may  be  re- 
marked here  that  the  vinegar  bacteria  cannot  attack  the  sugar  directly 
to  any  appreciable  degree,  and  the  manufacture  of  vinegar  from  sugar 
requires  two  agents,  the  alcohol-forming  yeast,  and  the  alcohol-oxidizing 
bacterium. 


PRODUCTS    OF   MICROBIAL  ACTIVITIES  239 

Some  of  the  acids  can  also  undergo  an  anaerobic  fermentation. 
This  is  possible  only  with  hydroxy-acids.  The  fermentation  of  the 
calcium  salt  of  tartaric  acid  was  the  first  anaerobic  fermentation 
observed  by  Pasteur,  and  the  fermentation  of  lactic  acid  to  butyric 
acid  has  a  reputation  for  its  chemical  peculiarity.  A  compound  with 
four  carbon  atoms  is  formed  from  a  compound  with  only  three  carbons, 
a  very  unusual  thing  in  fermentation. 

FATS. — The  decomposition  of  fats  is  comparatively  simple.  All  fats 
are  glycerides  of  organic  acids,  and  if  they  are  attacked  at  all  by  micro- 
organisms, they  are  first  split  into  glycerin  and  free  acid. 

H2C  -  O  -  CO  -  Ci5H31      HOH        H2COH      HO2C-Ci6H3i 

I  I 

HC  -  O  -  CO  -  Ci5H3i  +  HOH  =    HCOH  -f-  HO2C-Ci5H31 

.       I  I 

H2C  -  O  -  CO  -  CisHsi      HOH       H2COH      HO2CCi5H31 

Fat  Water  Glycerin  Acid 

This  brings  about  the  liberation  of  three  molecules  of  free  acid  from 
neutral  fat  molecule.  It  is  customary  to  test  for  the  splitting  of  fat  by 
determining  its  acidity.  The  glycerin  is  readily  used  up  by  the  micro- 
organisms, while  the  fatty  acids  are  oxidized  but  very  slowly. 

The  number  of  organisms  which  can  attack  fat  is  quite  small. 
Most  molds  can  destroy  it;  one  torula  has  been  found  in  butter  which 
attacks  it,  and  perhaps  a  dozen  species  of  bacteria  will  do  the  same, 
among  them  B.  fluorescens  and  B.  prodigiosus ,  which  cause  occasionally 
the  rancidity  of  butter. 

PRODUCTS  FROM  NITROGENOUS  COMPOUNDS 

On  account  of  the  complexity  of  the  protein  molecule,  the  products 
of  protein  decomposition  by  microorganisms  are  little  known.  Some 
products  are  conspicuous  through  their  odor,  others  can  be  told  by  cer- 
tain color  reactions,  but  as  we  cannot,  at  the  present,  give  the  structural 
formula  of  proteins,  there  is  no  possibility  of  stating  protein  decomposi- 
tions in  equations  similar  to  those  of  carbohydrate  fermentations. 
The  discussion  must  be  limited,  for  this  reason,  to  the  enumeration  of  the 
most  important  products,  and  to  the  general  types  of  decomposition. 

As  in  the  carbohydrates,  soluble  compounds  are  more  easily  de- 
composed than  the  insoluble.  The  keratin  bodies  of  hair,  epidermis 
and  horn  are  slowly  attacked  by  a  very  few  organisms.  Gelatin, 


240  NUTRITION    AND    METABOLISM 

casein  and  serum  albumin  are  more  readily  decomposed,  though  their 
solubility  is  quite  limited.  Peptones  which  are  readily  soluble  are 
used  by  the  vast  majority  of  microorganisms.  Of  interest  in  this  con- 
nection is  the  fact  that  the  fresh  white  of  egg  is  poisonous  to  most  bac- 
teria, and  fresh  blood  and  animal  tissues  as  well  as  freshly  drawn  milk 
have  also  germicidal  properties  which  are  lost  by  heating  or  upon 
standing. 

PROTEIN  BODIES  are  as  numerous  as  plants  and  animals.  Each 
species  of  organism  seems  to  have  its  particular  protein  which  differs 
from  that  of  other  species.  With  the  more  highly  developed  organisms, 
there  are  several  distinctly  different  proteins  found  in  the  same  individ- 
ual in  different  parts  of  the  body.  The  constituents,  carbon,  oxygen, 
hydrogen,  nitrogen,  and  sometimes  sulphur  and  phosphorus  can  be 
determined  in  their  relative  amounts  without,  however,  furnishing  any 
knowledge  of  the  structure  of  the  molecule.  The  molecular  weight  of 
proteins  is  estimated  to  be  at  least  10,000,  while  the  weight  of  the  very 
large  molecule  of  saccharose  is  only  342.  The  protein  molecule  can  be 
broken  up  into  smaller  molecules.  This  cleavage  is  generally  believed 
to  be  a  hydrolytic  process  similar  to  the  decomposition  of  starch  to 
maltose.  The  first  products  of  protein  decomposition  do  not  differ 
essentially  from  the  original  protein,  but  they  can  be  hydrolyzed  again 
and  again,  until  finally  products  of  a  crystalline  nature  are  found  which 
are  well-defined  chemical  bodies.  Among  the  very  first  products  of 
protein  degradation  it  is  usually  impossible  to  determine  single  com- 
pounds, but  several  groups  of  compounds  may  be  separated  by  certain 
precipitants,  as  acetic  acid,  ammonium  sulphate,  zinc  sulphate,  copper 
sulphate,  tannic  acid  and  others.  In  order  to  determine  the  degree  of 
protein  degradation,  e.g.,  in  the  analysis  of  cheese,  it  is  customary  to 
determine  the  nitrogen  of  compounds  precipitated  by  these  various 
reagents,  and  state  it  in  percentage  of  the  total  nitrogen.  Thus  the 
terms  "water-soluble  nitrogen,"  "acid-soluble  nitrogen"  and  others 
originated,  meaning  the  nitrogen  of  the  compounds  soluble  in  water  or 
in  acid  respectively.  Some  of  these  groups  of  degradation  products 
have  been  named  and  defined  more  accurately,  of  which  the  albumoses 
and  peptones  are  the  most  common  and  best  described  compounds. 
Their  chemical  nature  and  structure  is,  however,  just  as  little  known  as 
that  of  the  protein  bodies.  We  speak  of  peptonisation  of  proteins, 


PRODUCTS    OF    MICROBIAL   ACTIVITIES  241 

e.g.,  in  the  clearing  of  milk  or  the  gelatin  liquefaction,  meaning  that  the 
insoluble  protein  has  been  made  soluble. 

The  amino-acids  are  the  first  well  known  compounds  of  protein  de- 
composition. They  are  organic  acids,  in  which  a  hydrogen  atom  is 
substituted  by  a  NH2  radical.  Some  of  them  are  simple  compounds, 
as  the  amino-acetic  acid  NH2CH2COOH  and  also  the  amino-capronic 
acid  usually  called  leucin  (CH3)2CH  CH2  CH(NH2)  COOH.  Others 
are  of  a  more  complex  nature,  such  as  the  tyrosin  or  hydroxy-phenyl- 
aminopropionic  acid,  C6H4(OH)  CH2CH  (NH2)  COOH,  and  the 
tryptophan  or  indol-amino-propionic  acid,  C8H6N  CH2CH(NH2) 
COOH. 

Of  other  nitrogenous  products  which  are  not  amino-acids,  a  few 
are  of  striking  significance.  The  very  disagreeable  odor  of  putrefying 
proteins  and  of  excreta  is  due  to  indol  (C8H7N)  and  methyl-indol  or 
skatol  (C8H6N-CH3).  Indol  gives  a  rose  color  with  nitrites  in  acid 
solution,  and  this  convenient  reagent  is  used  in  the  identification  of 
bacteria.  Another  group  are  the  amins.  The  simplest  amins  are 
the  methyl-amins,  of  which  the  tri-methylamin  (CH3)3N  is  produced 
by  several  bacteria.  The  fishy  odor  of  the  brine  of  salted  herring  is 
largely  due  to  this  compound.  In  this  group  belong  also  a  large  number 
of  the  so-called  ptomains. 

The  ptomains  (page  592)  are  alkaloid-like  bodies  of  basic  character 
and  of  more  or  less  well-known  structure.  Some  of  them  are  notorious 
for  being  very  strong  poisons,  while  others  are  quite  harmless.  These 
bodies  are  called  ptomains  because  they  were  first  discovered  in 
putrefying  corpses.  The  best-known  compounds  of  this  character 
are  the  putrescin  or  tetra-methylen  diamin  [NH2(CH2)4NH2]  and  the 
cadaverin  or  penta-methylen-diamin  [NH2(CH2)5NH2],  which  can 
scarcely  be  considered  poisonous.  The  methyl-guanidin 

NH2 
HN  =  C<^ 

NHCH3 

may  be  mentioned  as  an  example  of  a  very  poisonous  ptomain.  Another 
poisonous  ptomain  is  the  neurin  CH2  =  CH  —  N(CH3)3OH  which  has 
been  found  frequently  as  a  product  of  putrefaction. 

Ammonia  is  the  end  product  of  protein  decomposition,  as  far  as 

Hi 


242  NUTRITION   AND   METABOLISM 

the  nitrogen-containing  fragments  of  the  protein  molecule  are  con- 
cerned. That  ammonia  is  formed  by  many  microorganisms,  is  well 
known.  In  some  decaying  proteins,  e.g.,  in  old  Camembert  cheese, 
ammonia  can  be  very  easily  detected  by  the  smell.  As  all  proteins 
conta'n  many  amino-groups  as  well  as  ac'd-amid  groups,  it  is  easily 
understood  how  the  ammonia  originates  through  the  hydrolysis  of  pro- 
tein. In  the  complete  oxidation  of  proteins  the  nitrogen  is  always  left 
as  NH3  or  (NH4)2CO3,  respectively,  never,  so  far  as  known,  in  any  other 
form.  No  bacterium  is  known  to  produce  urea,  as  most  of  the  higher 
animals  do. 

In  the  products  of  protein  degradation  mentioned  above  only 
those  compounds  have  been  considered  which  contain  nitrogen.  It  is 
quite  evident,  however,  that  in  the  cleavage  of  the  large  and  complex 
protein  molecules,  certain  parts  of  the  molecule  will  contain  no  nitrogen. 
Many  organic  acids,  like  acetic,  butyric,  capronic,  benzoic  and  phenyl- 
acetic  acids  are  quite  generally  found  among  the  products  of  putre- 
faction. Alcohols  too,  especially  benzene  derivatives  like  phenol  and 
cresol,  are  not  unusual.  Gas  is  often  formed  in  putrefaction,  especially 
carbon  dioxide  and  hydrogen;  occasionally  these  gases  are  mixed  with 
traces  of  nitrogen  and  methane. 

Many  protein  compounds  contain,  besides  the  organic  elements, 
larger  or  smaller  amounts  of  phosphorus  and  sulphur.  The  phos- 
phorus compounds  may  be  changed  to  phosphine  (PH3),  which  is  a  gas 
of  a  strong  disagreeable  garlic  odor.  Generally,  however,  the  phos- 
phorus of  protein  after  its  degradation  is  found  as  phosphoric  acid 
(H3PO4).  Very  little  is  known  about  the  phosphorus  of  organic 
compounds  and  the  changes  it  may  undergo  in  the  putrefactive  process. 

The  sulphur  of  proteins  is  commonly  changed  to  hydrogen  sulphide 
(H2S).  Some  microorganisms  are  able  to  form  mercaptan  (CH3SH), 
a  compound  of  very  foul  penetrating  odor. 

After  this  enumeration  of  the  products,  the  main  types  may  be 
considered  briefly;  since  much  less  work  has  been  done  on  protein 
decomposition  than  on  carbohydrate  decomposition,  the  groups  are 
not  so  well  defined.  We  might  consider  the  following  types: 

Complete  Oxidation.— This  is  brought  about  by  many  molds,  by 
yeasts  if  they  depend  upon  proteins  only,  and  by  many  bacteria,  of 
which  the  large,  aerobic  spore-forming  rods,  such  as  B.  mycoides,  are 
the  main  representatives.  The  products  of  oxidation  are  CO2, 


PRODUCTS    OF   MICROBIAL   ACTIVITIES  243 

NH3  and  H2SO4.  The  nitrogen  is  never  changed  to  any  oxidation 
product,  but  is  found  as  NH3,  while  the  sulphur  is  oxidized. 

Incomplete  oxidation  is  caused  by  other  bacteria,  and  perhaps  molds 
and  yeasts.  Quite  a  large  number  of  organisms  live  on  sugar-free 
media  if  they  have  oxygen,  but  they  do  not  oxidize  their  food  com- 
pletely. We  can  distinguish  at  least  three  different  groups  of  micro- 
organisms here. 

B.  proteus  is  the  collective  name  for  a  number  of  closely  related 
forms  which  belong  to  the  most  common  organisms  found  on  decaying 
organic  matter,  especially  when  protein  is  abundant.  They  produce 
leucin,  tyrosin  and  tryptophane,  but  no  skatol,  or  phenol.  Tndol 
and  hydrogen  sulphide  are  formed  in  certain  media.  Less  important, 
but  also  very  common  are  the  pigment-forming  rods  among  which  B. 
fluorescent,  B.  prodigiosus,  Ps.  pyocyanea  are  the  best-known  repre- 
sentatives. Their  metabolism  is  a  little  different;  amins  and  ammonia 
are  formed,  while  hydrogen  sulphide,  phenol  and  indol  are  absent. 
As  a  third  group,  B.  coli  may  be  mentioned  which  forms  indol,  but  no 
ammonia  from  peptone,  and  whose  proteolytic  powers  are  very  weak 
as  it  does  not  even  liquefy  gelatin. 

Anaerobic  decomposition  of  proteins  is  limited  to  very  few  species; 
there  is  a  great  difference  in  the  availability  of  proteins  and  of  carbo- 
hydrates as  a  source  of  energy,  protein  being  available  only  to  a  few 
species,  most  of^these  preferring  carbohydrates  if  they  are  present 
together  with  protein.  B.  putrificus  is  the  main  representative,  but 
other  forms  exist.  B.  putrificus  is  strictly  anaerobic,  and  a  spore  former, 
very  common  in  nature.  Among  the  products  are  skatol,  hydrogen 
sulphide,  ammonia  and  other  very  offensive  compounds. 

UREA,  URIC  ACID,  HIPPURIC  ACID,  are  the  end  products  of  protein 
metabolism  of  the  higher  animals.  The  decomposition  of  urea  to 
ammonium  carbonate  has  been  mentioned  in  several  places,  mainly 
on  page  202.  It  is  a  simple  hydrolysis 

CO(NH2)2  +  2H20  =  (NH4)2C03. 

This  change  can  be  brought  about  by  only  a  few  bacteria  which  are 
commonly  grouped  together  as  "urea  bacteria."  These  organisms 
have  hardly  anything  else  in  common,  however,  and  the  group  is  not  a 
well-defined  one.  There  are  rods  and  coccus  forms,  motile  and  non- 
motile  organisms,  spore-formers  and  non-spore  formers,  and  even  molds 


244  NUTRITION   AND    METABOLISM 

have  recently  been  found  to  hydrolyze  urea.  All  urea  bacteria  can 
live  without  urea,  feeding  on  organic  matter  like  other  bacteria,  but 
most  of  them  require  an  alkaline  medium. 

Hippuric  acid  is  split  by  certain  bacteria  to  benzoic  acid  and 
amino-acetic  acid  which  can  be  oxidized  completely.  Uric  acid  can  be 
changed  in  several  ways.  In  some  of  these  changes,  urea  is  found  as 
an  intermediary  product. 

PRODUCTS  FROM  MINERAL  COMPOUNDS 

Minerals  are  used  freely  by  microorganisms  for  cell  construction, 
consequently,  they  do  not  leave  the  living  cell  like  fermentation 
products.  But  a  few  organisms  can  actually  decompose  mineral 
matter  and  when  this  takes  place  mineral  products  are  secreted.  Two 
main  processes  can  be  distinguished,  oxidation  and  reduction. 

OXIDATIONS  are  the  result  of  the  organisms  seeking  a  supply  of 
energy.  Several  oxidations  of  minerals  have  been  indicated  previously, 
as  the  oxidation  of  ammonia  to  nitrites,  of  nitrites  to  nitrates,  of  hypo- 
sulphites to  sulphates,  of  hydrogen  sulphide  to  sulphur  and  of  sul- 
phur to  sulphuric  acid,  of  ferrous  salts  to  ferric  salts.  All  these 
microbial  changes  are  .simple  processes  and  can  be  followed  by  chem- 
ical analysis  much  more  easily  than  organic  fermentations.  The 
organisms  which  cause  these  changes,  do  not,  as  a  rule,  thrive  in 
organic  substances  and  for  this  reason  pure  cultures  can  be  obtained 
only  with  difficulty.  Their  activity  is  of  great  importance  in  soil 
fertility. 

REDUCTIONS  of  minerals,  too,  are  of  great  significance.  As  a  typical 
example,  nitrates  may  be  reduced  to  nitrites,  to  ammonia,  to  nitrogen 
gas,  and,  rarely,  to  nitrogen  oxides.  The  reduction  may  be  performed 
either  by  the  direct  removal  of  oxygen,  or  by  the  formation  of  free 
oxygen.  The  reduction  of  nitrates  to  nitrites  can  be  written  in  the 
following  three  ways: 

KNO3  -     O  =  KNO2 
KNO3  =  KNO2  +  O 

KNO3  +  2H  =  KNO2  +  H2O. 

The  result  in  all  three  cases  is  the  same.  Many  bacteria  can  reduce 
nitrates  to  nitrites  or  to  ammonia.  A  few  can  reduce  them  to  nitrogen. 


PRODUCTS    OF    M1CROBIAL   ACTIVITIES  245 

These  "true  denitrifiers "  are  found  in  soil  and  in  old  manure.  Their 
reducing  process  is  as  follows: 

Ca(NO3)2  -  5O  =  CaO  +  2N. 

Nitrates  are  reduced  through  the  efforts  of  the  organism  to  secure  a 
supply  of  oxygen.  The  denitrifying  bacteria  have  strong  oxidizing 
properties;  they  take  oxygen  from  all  sources  possible.  If  cultures  of 
denitrifying  bacteria  are  well  aerated,  as  in  soils  with  a  proper  mois- 
ture content,  they  scarcely  attack  the  nitrates,  while  they  will  reduce 
them  in  ordinary  liquid  cultures  so  fast  that  the  escaping  nitrogen 
gas  forms  a  froth  on  top  of  the  nitrate  solution.  Denitrifying  bacteria 
need  the  oxygen  to  oxidize  organic  matter.  They  cannot  live  without 
organic  food. 

Sulphates  are  reduced  in  a  very  similar  way  to  hydrogen  sulphide 

H2SO4  -  40  =  H2S. 

Tap-water,  containing  calcium  sulphates,  often  forms  hydrogen  sulphide 
if  shut  off  from  the  air  for  some  time. 

While  only  a  few  bacteria  reduce  sulphates,  many  reduce  sulphites  or 
sulphur  to  hydrogen  sulphide.  The  potassium  and  sodium  salts  of 
selenic  and  telluric  acid  (H2SeO4  and  H2TeO4)  are  reduced  by  certain 
organisms  and  not  by  others.  The  reduction  results  in  a  colored 
precipitate;  this  reaction  has  been  suggested  as  a  diagnostic  means  to 
distinguish  different  species.  The  reduction  of  arsenious  oxide  to 
arsin  (AsH3)  is  used  as  a  very  delicate  test  for  arsenic;  it  is  applied  in 
the  detection  of  arsenical  poisoning.  The  material  to  be  tested  is 
sterilized  and  inoculated  with  Penicillium  brevicauk  (page  53,  the 
"arsenic  mold").  This  will  reduce  most  arsenious  compounds  to  arsin 
(AsH3)  or  to  diethyl  arsin,  AsH(C2H5)2,  both  of  which  are  easily 
recognized  by  their  very  pronounced  garlic  odor. 

UNKNOWN  PRODUCTS  OF  PHYSIOLOGICAL  SIGNIFICANCE 

Among  the  products  of  microbial  action,  there  are  certain  substances 
which  must  be  mentioned  because  of  their  importance,  though  their 
quantity  is  insignificant  compared  with  the  ordinary  products  of  fermen- 
tation. These  substances  can  be  divided  into  four  groups:  pigments, 
aromatic  compounds,  enzymes,  and  toxins.  The  chemical  structure  of 
pigments  and  of  many  aromatic  substances  is  scarcely  known;  and  as 


246  NUTRITION  AND   METABOLISM 

far  as  enzymes  and  toxins  are  concerned,  it  is  not  even  determined 
whether  or  not  they  are  of  protein  nature.  The  last  two  groups  are 
known  only  by  their  actions,  while  the  pigments  are  very  conspicuous 
and  cannot  possibly  be  overlooked. 

PIGMENTS  have  naturally  attracted  the  attention  of  microbiologists 
ever  since  pure  cultures  were  known,  and  many  investigators  have  tried 
to  explain  the  nature  and  the  meaning  of  pigments.  All  experiments 
concerning  the  purpose  of  pigment-formation  by  microorganisms  have 
been  without  results.  It  is  not  known  that  the  pigment  is  of  any 
material  advantage  to  bacteria;  for  it  is  possible  to  cultivate  colorless 
strains  of  pigment  bacteria  which  grow  apparently  as  well  as  the  original 
pigmented  culture.  Again,  pigments  cannot  take  the  place  of  the 
chlorophyl  in  plants  except  perhaps  the  bacteriopurpurin  of  the  purple 
bacteria.  It  does  not  even  protect  the  cells  against  intense  light, 
because  the  pigmented  organisms  are  not  more  resistant  than  the  corre- 
sponding colorless  "sports."  The  only  exception  are  the  colored  spores 
of  the  molds,  especially.  Penicillium  and  Aspergillus,  which  are  very 
resistant  to  light,  while  the  spores  of  Oidium  are  killed  just  as  easily  as 
the  mycelium.  Pigments  cannot  be  considered  as  reserve  substances, 
since  many  pigments  are  excreted  and  remain  outside  the  colorless 
cells.  Pigment  production  may  be  incidental.  It  is  possible  that  the 
waste  products  of  certain  organisms  happen  to  be  colored. 

After  Beyerinck,  the  chromogenic  bacteria  may  be  divided  into  three 
classes : 

1.  Chromophorous  bacteria,  in  which  the  pigment  is  placed  in  the  cell 
and  has  a  certain  biological  significance  analogous  to  the  chlorophyl 
of  higher  plants.     In  this  division  belong  the  green  bacteria  discovered 
by  Van  Tieghem  and  Engelmann  and  the  red  sulphur  bacteria  or  purple 
bacteria. 

2.  Chromoparous  or  true  pigment-forming  bacteria,  which  set  free  the 
pigment  as  a  useless  excretion,  either  as  a  color-body  or  as  a  leuco-body 
which  becomes  colored  through  the  action  of  atmospheric  oxygen.     The 
individuals  themselves  are  colorless  and  may  under  certain  conditions 
cease  to  form  pigments.     To  this  class  belong  B.  prodigiosus,  B.  cyano- 
genes,  Ps.  pyocyanea,  and  others. 

3.  Parachrome  bacteria,  which  form  the  pigment  as  an  excretory  prod- 
uct but  retain  it  within  their  bodies,  as  B.  janthinus  and  B.  violaceus. 

When  the  pigment  is  soluble  in  water,  as  those  produced  by  Ps, 


PRODUCTS    OF   MICROBIAL  ACTIVITIEP  247 

pyocyanea  and  the  fluorescent  bacteria,  it  diffuses  through  the  medium. 
When  the  pigment  is  not  soluble,  it  either  lies  within  the  cell  wall  or 
between  the  individuals. 

This  classification  furnishes  some  details  concerning  the  methods  of 
pigment  production,  which  depends  upon  the  presence  of  certain  media. 
According  to  Sullivan,  sometimes  certain  mineral  salts,  sometimes  sugar 
will  stimulate  chromogenesis.  The  same  is  true  with  molds.  Very 
brilliant  colors  appear  with  certain  species  of  molds  if  grown  on  cellu- 
lose or  on  fat,  while  on  gelatin  the  pigment  is  not  produced.  The  tem- 
perature is  an  important  factor.  A  large  number  of  chromogens 
produce  no  pigment  when  grown  in  the  incubator.  It  is  possible  to 
obtain  non-pigmentation  with  many  species  by  propagating  them 


FIG.  113.— Bacteriopurpin,  from  a  Rhodes pir ilium,  crystallized  from  a  chloroform 
solution.     (After  Molisch.) 

through  many  generations  at  high  temperatures.  Oxygen  also  is 
necessary  for  the  chromogenesis  of  many  bacteria.  Some  need  a  short 
exposure  to  daylight  in  order  to  produce  their  pigment,  while  cultures 
grown  in  absolute  darkness  may  remain  colorless.  Strong  sunlight, 
however,  will  check  pigment  production  in  the  same  degree  as  do 
antiseptics  and  other  harmful  influences. 

The  chemical  nature  of  microbial  pigments  is  little  known.  They 
are  distinguished  according  to  the  solubility  in  various  liquids,  as  water, 
alcohol,  ether,  chloroform,  benzol,  and  other  solvents,  and  according 
to  the  change  of  color  caused  by  acid  and  alkali.  A  group  of 
carotin  bodies,  named  because  of  their  similarity  to  the  pigment 
of  carrots,  the  prodigiosin  bodies,  named  after  B.  prodigiosus,  the 


248  NUTRITION   AND    METABOLISM 

fluorescent  pigments  and  perhaps  a  few  other  groups  are  distinguished, 
but  their  chemical  nature  is  rather  vague  as  yet.  The  absorption  of 
distinct  lines  of  the  spectrum  by  solutions  of  these  pigments  is  claimed 
to  be  a  very  reliable  means  of  distinguishing  the  pigments  of  different 
species. 

AROMATIC  SUBSTANCES  constitute  another  group  of  metabolic  prod- 
ucts. The  chemical  analysis  accomplishes  more  with  these  com- 
pounds than  with  pigments,  since  they  are  frequently  well-known 
compounds.  The  main  difficulty  arising  in  their  identification  is  in 
the  very  minute  quantities  of  the  products  available.  Some  substances 
with  strong,  mostly  very  disagreeable  odors  have  already  been  men- 
tioned: indol,  skatol,  hydrogen  sulphide,  mercaptan,  the  amins  and 
ammonia,  butyric  acid,  and  some  of  the  higher  alcohols.  There  re- 
main to  be  mentioned  certain  oils  and  esters  giving  rise  largely  to 
pleasant  aromas.  The  formation  of  aromatic  oils  has  been  established 
although  their  nature  is  entirely  unknown.  The  same  is  true  with  the 
esters.  The  substance  causing  the  fishy  flavor  in  butter  is  volatile 
with  steam  and  is  neither  of  an  alkaline  nor  acid  nature.  The  strong 
odor  of  freshly  plowed  earth  is  caused  by  an  Actinomyces;  the  odor 
can  be  traced  to  a  very  volatile  oil  the  nature  of  which  has  not  been 
determined.  The  aroma  of  fermented  liquids — wines,  beers,  and 
many  others — is  partly  due  to  compounds  constituting  the  fermenting 
material,  and  partly  to  the  fermenting  agent.  Some  yeasts  are 
known  to  produce  fruit-esters,  as  succinic-acid-ethylester  and  the 
corresponding  esters  of  malic  and  other  acids.  Besides,  some  glucosides 
may  be  split  and  traces  of  hydrocyanic  acid  and  benzoic  acid  may  be 
liberated.  The  change  of  flavor  with  the  aging  of  wines  is  probably 
more  a  chemical  than  a  biochemical  change. 

ENZYMES  AND  TOXINS. — Among  the  most  interesting  and  least 
understood  products  of  microbial  action  are  the  enzymes  and  the  toxins. 
These  two  groups  are  related  in  many  respects  The  enzymes  have 
been  discussed  extensively  in  a  preceding  chapter  and  toxins  will  be 
treated  more  extensively  on  pages  676,  740.  Toxins  and  enzymes  are 
formed  by  the  cells  in  such  small  quantities  that  they  would  never  have 
been  discovered  by  ordinary  chemical  means  were  it  not  for  the  unusual 
effects  which  they  produce,  the  enzymes  acting  upon  food  substances, 
and  the  toxins  acting  physiologically  upon  organisms.  Toxins  and 
enzymes  are  chemically  unknown.  It  is  assumed  that  they  are  chemical 


PRODUCTS    OF    MICROBIAL   ACTIVITIES  249 

bodies,  but  even  this  has  not  been  proved.  A  pure  toxin  has  never 
been  obtained  and  we  have  no  criterion  for  its  purity.  The  presence 
of  a  toxin  is  recognized  only  by  an  animal  test  and  in  this  way  the  com- 
parative concentration  can  be  determined  approximately.  Such 
standardization  of  toxin  solutions  is  only  comparative,  however,  and 
gives  no  clue  as  to  the  actual  amount  of  toxin  present.  Not  all  ani- 
mals are  sensitive  to  all  toxins.  It  is  quite  possible  that  all  bacteria 
produce  compounds  with  chemical  qualities  similar  to  toxins,  and  only 
a  few  of  them  happen  to  react  upon  men  or  animals. 

Toxins  are  not  always  the  product  of  microbial  action.  Vegetable 
toxins  or  phytotoxins  are  known,  among  which  the  ricin  of  the  castor- 
oil  bean  is  perhaps  the  most  studied  representative.  The  best-known 
zootoxin  is  the  rattlesnake  poison.  These  non-microbial  compounds 
have  the  same  quality  as  the  microbial  toxins — they  are  extremely 
poisonous.  Toxins  are  the  cause  of  disease  in  diphtheria,  tetanus  and 
botulism.  If  a  culture  of  these  organisms  is  filtered  through  a  porcelain 
filter  which  removes  all  bacterial  cells,  the  filtrate  injected  into  an 
animal  will  cause  the  disease  with  all  its  accompanying  symptoms 
though  there  are  no  microorganisms  introduced  into  the  animal  body. 
If  the  filtrate  is  heated,  however,  no  effect  will  take  place  after  the  in- 
jection, because  heat  destroys  the  toxin.  The  amount  of  toxin  that  will 
kill  an  animal  is  extremely  small.  .000005  mg-  °f  the  purest  tetanus 
toxin  will  kill  a  mouse,  .0007  mg.  of  ricin  will  kill  a  rabbit,  less  than 
.23  mg.  of  tetanus  toxin  will  kill  an  adult  man.  The  body  of  an  animal 
,or  man  forms  an  anti-body  against  the  toxin  which  neutralizes  its 
poisonous  action.  Anti-bodies  are  also  formed  against  enzymes 
injected  into  an  animal. 

Toxins  are  very  sensitive  to  heat.  A  short  exposure  to  temperatures 
between  80°  and  100°  will  inactivate  them.  They  are  also  very  sensi- 
tive to  light.  While  some  toxins  are  secreted,  others  are  retained  within 
the  cells  of  microorganisms,  and  never  leave  them  until  the  cells  die  or 
disintegrate.  Ptomains,  which  are  also  metabolic  products  of  micro- 
organisms and  sometimes  cause  poisoning,  differ  from  the  toxins  in  their 
resistance  to  heat  and  light  (page  241).  Ptomains  differ  in  no  way 
essentially  from  ordinary  organic  compounds;  the  animal  or  human 
body  produces  no  anti-ptomains  to  counteract  their  poisonous  effects. 
There  is  no  chemical  relation  whatever  between  toxins  and  ptomains, 


250  NUTRITION   AND    METABOLISM 

and  the  physiological  effects  are  also  quite  different,  though  they  both 
cause  poisoning. 

Toxins  are  not  essential  products  of  the  metabolism  of  pathogens. 
Strains  of  pathogenic  bacteria  can  be  bred  which  do  not  produce  toxins 
as  chromogens  can  be  bred  without  pigment,  or  lactic  bacteria  which 
do  not  produce  acid.  The  strains  which  lose  their  pathogenicity  grow 
better  on  artificial  media  but  are  less  able  to  produce  disease  in  the 
animal.  They  may  regain  the  power  of  producing  toxin  if  passed 
through  the  body  of  the  animal.  The  real  object  of  toxin  production 
by  microorganisms  is  not  known;  the  microorganisms  derive  no  ap- 
parent benefit. 

PHYSICAL  PRODUCTS  or  METABOLISM 

PRODUCTION  OF  HEAT. — It  has  long  been  known  that  fermentation 
produces  heat.  The  rise  of  temperature  is  usually  not  very  great.  In 
lactic  fermentation  it  amounts  to  about  i°,  in  alcoholic  fermentation  to 
2  or  3°,  but  in  certain  processes  the  heat  liberated  is  considerable,  as 
in  the  fermentation  of  manure,  of  ensilage,  of  vinegar,  and  in  others. 

The  cause  of  heat  formation  is  quite  evident  from  the  discussion  on 
page  199.  Decomposition  of  organic  matter  means  a  liberation  of 
energy  which  is  used  for  the  continuation  of  life  processes;  the  utiliza- 
tion is,  as  a  rule,  incomplete,  and  a  part  of  the  energy  appears  in  the 
form  of  heat.  The  amount  of  heat  produced  can  be  measured  directly 
with  the  thermometer  if  great  care  is  taken  that  no  heat  is  lost  by 
radiation  or  by  evaporation  of  water. 

Much  heat  is  produced  in  the  vinegar  fermentation.  In  the  quick- 
vinegar  process  (page  644)  the  temperature  rises  sometimes  as  high  as 
10°  to  15°  above  the  temperature  of  the  room  and  the  vinegar  manu- 
facturer uses  the  heat  produced  by  the  bacteria  to  keep  the  generators 
at  the  optimum  temperature.  If  the  process  is  not  controlled  carefully, 
the  vinegar  bacteria  are  likely  to  produce  sufficient  heat  to  kill 
themselves. 

The  heat  produced  in  the  fermentation  of  manure,  especially  horse 
manure,  is  used  in  the  hot-beds  to  cultivate  and  force  young  plants. 
In  the  manure  pile,  great  heat  production  is  not  desirable  because  high 
temperatures  will  volatilize  the  ammonia;  the  tight  packing  of  manure 
which  keeps  out  the  oxygen  will  prevent  too  strong  bacterial  action. 
The  highest  temperature  in  silos  which  has  been  recorded  is  about  70°, 


PRODUCTS    OF   MICROBIAL   ACTIVITIES  25! 

but  the  best  silage  is  secured  by  keeping  the  temperature  below  50°. 
Ensilage  fermentation  is  not  thoroughly  understood,  however,  and  no 
accurate  statements  can  be  made  as  to  the  cause  of  the  increase  in 
temperature.  Sometimes  the  temperature  in  silos  does  not  exceed 
35°.  The  curing  of  hay  is  usually  accompanied  by  a  rise  of  temperature. 
For  some  time  it  was  believed  that  the  spontaneous  combustion  of  hay 
was  mainly  due  to  microorganisms,  but  it  has  been  shown  recently 
that  even  sterile  hay  will  show  a  rise  of  temperature  under  certain 
conditions.  This  does  not  exclude  the  formation  of  heat  in  hay  by 
microorganisms  under  other  circumstances.  The  heating  of  tobacco, 
of  green  or  moist  grain  or  corn  is  not  of  bacterial  origin,  but  due  to 
the  respiration  of  the  living  plant-tissue. 

PRODUCTION  OF  LIGHT. — The  light-producing  or  photogenic  organ- 
isms are  quite  numerous  and  occur  more  frequently  than  is  generally 
believed.  The  phosphorescence  of  decaying  tree  stumps  and  leaves  in 
the  woods  and  of  meat  and  fish  in  the  cellar  are  well-known  phenomena. 
The  phosphorescence  of  wood  and  leaves  is  generally  caused  by  Hypho- 
mycetes;  certain  mushrooms  have  this  quality  in  a  very  high  degree. 
The  light  of  meat  and  fish  is  usually  generated  by  bacteria,  of  which  at 
least  twenty-six  species  have  been  described. 

"  The  most  obvious  evidence  of  liberation  of  energy  in  the  physiology 
of  protozoa  is  seen  in  their  movement.  Certain  protozoa,  Noctiluca 
for  example,  however,  emit  light  and  produce  the  phosphorescence 
often  observed  in  sea  water.  From  analogy  with  higher  animals  it  is 
to  be  supposed  that  heat  and  electrical  changes  are  also  produced." 

Many  experiments  have  been  carried  on  in  order  to  discover  the 
nature  and  origin  of  the  light,  but,  so  far,  few  results  have  been  obtained. 
The  phosphorescence  is  due  to  an  oxidation  process;  all  photogenic 
organisms  cease  to  generate  light  when  the  oxygen  is  removed.  As 
soon  as  they  come  into  contact  with  oxygen  again,  they  produce  light 
immediately,  and  this  sudden  flashing  is  used  occasionally  by  physiolo- 
gists as  a  very  delicate  test  for  oxygen.  The  light  appears  to  be  pro- 
duced always  within  the  cell;  no  cell  product  has  ever  been  found  to 
give  rise  to  light  outside  the  cell.  It  is  possible  that  a  chemical  com- 
pound is  formed  in  the  cell  which  generates  light  when  in  contact 
with  oxygen. 

The  life  processes  of  the  photogenic  microorganisms  are  not  neces- 
sarily connected  with  the  formation  of  light.  Photogenic  bacteria  are 


252  NUTRITION   AND    METABOLISM 

known  to  lose  the  power  of  light  production  as  the  chromogenic  bacteria 
may  lose  the  power  of  pigment  production.  Phosphorescence  has,  like 
pigmentation  also,  no  bearing  upon  the  development  of  the  cell,  and  the 
light-giving  compounds  may  be  regarded  as  incidental  waste  products. 
Certain  chemical  bodies  stimulate  light  production,  while  others  favor 
the  growth  only.  One  of  the  most  important  factors  in  the  production 
of  light  is  sodium  chloride. 


CHAPTER  V 

PHYSIOLOGICAL    VARIATIONS    ASSOCIATED    WITH    METABOLISM  AND 

NUTRITION  * 

The  great  variability  of  microorganisms  in  morphological  respects 
has  already  been  pointed  out  in  Part  I  of  this  book.  A  similar  variation 
and  adaptation  are  noticed  in  their  physiology,  especially  with  the  food 
substances  of  bacteria  and  consequently  with  their  metabolic  products. 
Microorganisms  change  their  physiological  properties  very  readily  with 
the  environment;  the  new  variety  may  keep  its  acquired  properties  for 
some  time  even  if  brought  back  to  the  original  conditions.  It  is  stated 
frequently  that  microorganisms  tend  more  toward  variations  than  the 
more  complex  organisms.  It  should  be  considered,  however,  that  the 
experiences  in  the  variations  of  green  plants  and  animals  are  based  on 
individuals,  while  in  the  case  of  microorganisms  these  experiences  are 
gained  almost  always  from  millions  of  cells.  A  simple  illustration  is  the 
development  of  bacteria  in  salt  solutions.  If  a  broth  culture  of  B.  coli 
is  transferred  into  broth  containing  8  per  cent  of  salt,  a  large  number  of 
cells  will  die,  often  more  than  99  per  cent.  The  surviving  bacteria  begin 
to  multiply  after  a  certain  length  of  time  and  a  new  variety  is  created 
which  can  tolerate  the  salt.  At  first,  only  about  one  out  of  one  hundred 
cells  had  the  power  to  tolerate  salt,  but,  since  the  dying  cells  are  not 
usually  counted  or  considered  at  all,  it  is  customary  to  say  that  bacteria 
easily  adapt  themselves  to  an  8  per  cent  salt  solution.  If  only  one 
single  plant  out  of  one  hundred  could  be  adapted  to  a  certain  high 
temperature,  it  could  not  be  said  that  it  adapts  itself  easily.  This  mis- 
take is  quite  commonly  made  with  microorganisms. 

The  best  illustration  for  the  variability  of  cultivated  microorganisms 
is  the  enormous  number  of  varieties  of  Saccharomyces  cerevisice.  Nearly 
every  large  brewery  has  a  yeast  type  of  its  own  which  differs  from  others 
by  the  amount  of  alcohol  and  aromatic  substances  produced,  by  time 
and  optimum  temperature  of  spore-production,  by  the  appearance  of 
the  budding  yeast  in  the  hanging  drop,  and  also  in  other  respects.  The 

*  Prepared  by  Otto  Rahn. 

253 


254  NUTRITION  AND   METABOLISM 

cultivated  organisms  are  not  alone  in  showing  this  tendency  toward 
variation.  The  transferring  of  a  soil  or  water  bacterium  into  the  ordi- 
nary laboratory  media  is  a  complete  change  of  conditions;  the  different 
cells  of  the  same  species  may  react  differently  and  give  several  varie- 
ties. A  lactic  bacterium  on  meat  medium  without  sugar  does  not  thrive 
well  in  the  first  generations,  but  it  gradually  becomes  able  to  grow  on 
this  medium.  By  this  treatment,  it  loses  gradually  the  power  of  pro- 
ducing acid  and  does  not  thrive  as  well  in  milk.  The  attenuation  of 
pathogenic  bacteria  by  cultivation  on  media,  as  potato,  very  different 
from  the  blood  and  muscle  upon  which  they  grow  most  naturally,  or 
by  growing  them  at  low  temperature,  or  above  the  maximum,  furnishes 
another  example.  The  decrease  and  finally  the  entire  loss  of  patho- 
genicity  is  caused  by  a  change  of  metabolism,  by  a  loss  of  the  power  to 
produce  toxin. 

As  by  certain  diet  the  metabolism  can  be  changed,  so  certain 
physiological  properties  of  bacteria  can,  by  proper  cultivation,  be 
increased.  By  the  frequent  transferring  of  an  organism  on  gelatin,  its 
liquefying  qualities  can  be  increased,  provided  it  had  some  at  the  start. 
By  continued  passing  of  a  bacterium  through  an  animal,  its  virulence 
can  be  increased.  Strains  of  bacteria  which  will  produce  a  very  high 
acidity  can  be  bred;  this  is  illustrated  by  the  quick- vinegar  process 
and  by  the  strong  alcohol-producing  yeasts  of  the  distillery  process. 
By  continued  cultivation  of  an  organism  upon  a  certain  medium,  it 
will  become  so  acclimatized  that  it  degenerates  readily  when  the  con- 
ditions become  unfavorable  Such  specifically  trained  strains  of 
microorganisms  are  used  in  alcoholic  and  lactic  fermentation,  in  patho- 
genic bacteriology  and  in  the  inoculation  of  leguminous  plants  with 
nitrogen-fixing  bacteria. 

FACTORS  INFLUENCING  THE  TYPE  OF  DECOMPOSITION 

In  the  chapter  on  products  of  metabolism,  it  has  been  shown 
that  the  same  compound  can  be  decomposed  in  many  different  ways, 
and  the  question  may  well  be  asked  what  decides  the  type  of  decomposi- 
tion. Since  bacteria  are  widely  distributed,  it  must  be  expected  that 
there  are  certain  conditions  which  are  most  favorable  to  a  given  type 
of  fermentation,  while  under  changed  conditions,  other  types  are  more 
likely  to  dominate.  The  fact  that  sugar  in  cider  nearly  always  under- 
goes alcoholic  fermentation,  while  in  milk  it  undergoes  lactic  fermen- 


PHYSIOLOGICAL  VARIATIONS  255 

tatioa,  has  its  reason  in  the  physiology  of  the  bacteria,  and  in  their 
reaction  upon  the  environment. 

Cider  is  acid,  and  acid  is  not  well  suited  for  the  growth  of  most 
bacteria.  The  vinegar  bacteria  can  grow  in  fruit  juices,  and  a  few  other 
bacteria,  especially  those  causing  trouble  in  wine,  are  not  retarded  by 
fruit  acids,  but  the  common  types  attacking  proteins  and  causing 
organic  decay  are  not  able  to  grow  on  fruits.  Yeasts,  however,  and 
molds  thrive  well  only  in  acid  media.  They  can  exist  in  neutral 
solutions  if  in  pure  culture,  but  in  nature  they  are  easily  crowded  out 
by  bacteria.  Acidity  of  the  medium  is  therefore  one  of  the  most 
important  factors  regulating  the  type  of  microbial  decomposition. 
This  principle  is  commonly  utilized  by  preserving  foods  of  all  kinds  in 
vinegar,  and  by  making  butter  from  sour  cream  rather  than  sweet 
cream;  the  keeping  qualities  of  hard  cheeses  depend  upon  their  acid 
content. 

In  acid  environment,  the  two  most  common  types  of  decomposition 
are  oxidation,  complete  or  incomplete,  and  alcoholic  fermentation. 
The  oxidation  is  brought  about  by  molds  or  organisms  closely  allied  to 
yeasts.  The  latter  are  very  common  on  all  sour  foods,  especially  on 
foods  containing  lactic  acid,  such  as  cottage  cheese  or  sauerkraut. 
The  kind  of  acid  decides  the  type  of  mold;  wherever  there  is  lactic  acid, 
there  is  Oidium,  while  malic  and  tartaric  acids  favor  Pemcillium  and 
A  spergillus. 

If  the  decaying  materials  contain  no  acid,  the  type  of  decomposi- 
tion depends  mainly  on  the  presence  or  absence  of  carbohydrates, 
especially  sugar.  It  is  an  old  experience,  recently  verified  through 
a  large  number  of  experiments  by  Kendall  and  Walker,  that  practically 
all  bacteria  will  decompose  sugar  in  preference  to  proteins.  If  a  leaf 
contains  sugar  and  protein  (cabbage)  the  sugar  decomposition  will  be 
conspicuous,  and  the  protein  is  not  attacked  very  readily.  Putrefac- 
tion in  the  presence  of  sugar  or  of  acid  does  not  take  place.  Meat  will 
not  putrefy  if  mixed  with  sugar,  while  milk  putrefies  readily  if  the  sugar 
is  removed  by  dialysis.  The  three  types  of  sugar  decomposition  which 
come  into  consideration  in  neutral  media,  are  the  lactic,  the  acid-gas  and 
the  butyric  fermentations.  The  latter  is  a  strictly  anaerobic  fermenta- 
tion, and  thus  limited  to  special  conditions.  Of  the  other  two,  the  acid- 
gas  fermentation  is  the  most  common,  and  the  souring  of  vegetables 
of  all  kinds  is  due  to  this  type  of  fermentation  (pickles,  sauerkraut, 


256  NUTRITION   AND    METABOLISM 

ensilage,  salt-rising  bread).  Sometimes  the  acid-gas  fermentation 
is  followed  by  a  butyric  fermentation.  The  true  lactic  fermentation 
is  not  common,  and  is  limited  almost  entirely  to  milk.  This  is  ex- 
plained by  the  circumstance  that  the  organisms  causing  this  decom- 
position are  parasitic  in  their  habits,  causing  "disease  or  living  in  the 
intestine  of  animals.  In  the  absence  of  acid  and  sugars,  putrefaction 
is  the  most  common  type  of  decomposition. 

Many  factors  aside  from  the  chemical  composition  of  the  medium 
are  essential.  Oxygen  has  already  been  mentioned  as  preventing  buty- 
ric fermentation.  It  will  also  prevent  the  acid-gas  fermentation  if  too 
abundant.  Ensilage  is  trampled  and  pressed  down  to  avoid  air  spaces 
as  much  as  possible,  for  molds  will  outgrow  the  acid-forming  bacteria 
if  air  has  free  access.  Absence  of  oxygen  will  prevent  mold  growth, 
and  for  this  reason,  jelly  is  paraffined,  and  butter  wrapped  tightly  into 
impermeable  paper.  The  influence  of  oxygen  upon  the  type  of  protein 
and  of  cellulose  decomposition  has  been  pointed  out  previously. 

The  moisture  content  is  of  great  importance.  As  will  be  shown 
later,  not  all  organisms  have  an  equal  need  of  moisture;  some  molds 
will  grow  on  foods  too  dry  for  bacteria  and  yeasts.  Molds  are  es- 
pecially adapted  for  growing  on  dry  media,  as  only  part  of  their  cell 
substance  is  immersed  in  the  medium.  Their  thread  formation  enables 
them  to  search  a  dry  medium,  such  as  flour,  for  moisture,  the  extreme 
of  adaptation  being  Rhizopus,  and  the  construction  of  the  fruiting 
bodies  shows  that  they  are  destined  by  nature  to  be  spread  by  air  and 
wind.  It  is  no  wonder  that  damp  organic  matter,  if  it  can  be  de- 
composed at  all,  will  show  molds,  and  nothing  else,  regardless  of  the 
chemical  composition,  for  there  is  no  competition.  Flour,  moist  seeds, 
incompletely  dried  fruit,  damp  milk  powder  will  always  become 
moldy.  The  same  holds  true  with  very  concentrated  sugar  solutions 
such  as  syrups,  jellies  and  jams,  while  in  concentrated  salt  solutions, 
molds  cannot  thrive,  and  the  torula  yeasts  are  best  adapted  to  such 
conditions. 

A  very  important  part  is  also  the  structure  of  the  material.  Micro- 
organisms act  mainly  upon  organic  matter,  and  since  this  comes 
from  living  organisms,  it  has  usually  definite  structure,  exceptions 
being  milk  and  blood.  The  structure  of  all  living  organisms  is  such 
as  to  prevent  the  intruding  of  microorganisms.  The  body  of  plants 
and  animals  is  surrounded  on  the  outside  by  tough  and  dry  layers  of 


PHYSIOLOGICAL  VARIATIONS  257 

epithelial  cells,  and  the  cavities  of  the  animal  body  also  have  their 
protective  membranes.  Microorganisms  cannot  enter  the  tissues 
if  these  membranes  are  perfectly  sound,  and  we  know  that,  as  a  rule, 
the  tissues  of  healthy  plants  or  animals  are  free  from  bacteria.  Thus, 
a  healthy  apple  or  potato  or  egg  will  not  be  infected  and  decomposed 
by  microorganisms  if  handled  carefully,  meat  will  begin  to  decom- 
pose on  the  outside,  and  the  inner  parts  may  be  still  good  when  the 
outer  layer  is  already  in  a  state  of  decay. 

In  the  plants,  each  cell  is  surrounded  by  its  special  cell  membranes 
which  are  a  barrier  to  infecting  organisms.  If  we  prick  the  skin  of  a 
healthy  apple  with  a  pin  infected  with  yeast,  the  infection  will  not 
spread  though  we  know  that  yeast  will  grow  most  abundantly  in  cider; 
in  the  apple,  however,  it  has  no  means  of  spreading  from  one  cell  to 
the  other.  Molds  possess  this  means;  they  can  puncture  cell  walls, 
and  forcing  their  way  from  one  cell  to  the  other,  they  will  soon 
bring  about  the  rotting  of  the  entire  fruit  after  it  once  becomes 
infected.  This  protection  seems  especially  necessary  in  the  plant's 
roots  which  are  greatly  exposed  to  injury  from  insects  and  other  animals 
in  the  soil  and  surrounded  by  billions  of  microorganisms.  They  are 
attacked  only  by  fungi  which  can  force  their  way  from  cell  to  cell, 
or  by  bacteria  which  can  dissolve  the  membranes  by  means  of  enzymes, 
and  thus  cause  a  softening  of  the  root  tissue.  The  bacteria  causing  the 
various  rots  of  vegetables  belong  to  this  type. 

There  is,  then,  a  great  variety  of  factors  deciding  the  type  of 
decomposition  of  organic  matter  in  nature,  and  by  knowing  the  chemical 
composition  as  well  as  the  structure  and  other  physical  conditions, 
it  is  possible  to  foretell  which  group  of  organisms  is  most  likely  to 
attack  the  compounds  in  question. 

Another  quite  important  factor,  the  temperature,  will  be  dis- 
cussed in  more  detail  in  one  of  the  following  chapters. 


17 


CHAPTER  VI 

NUTRIT  ION  OF  MICROORGANISMS  AND  THE  ROTATION  OF  ELEMENTS 

IN  NATURE* 

All  organic  matter  on  earth  is  undergoing  continuous  change.  Or- 
ganisms grow  and  decay.  The  same  carbon  and  nitrogen  atoms  which 
constitute  the  organic  world  of  to-day  constituted  it  thousands  of 
years  ago.  The  amount  of  carbon,  nitrogen,  hydrogen  and  of  all 
other  elements  of  life  on  earth  is  limited,  and  the  same  atoms  will 
be  used  for  the  future  generations  of  life  that  constitute  the  present. 
There  must  be  continuous  destruction  to  enable  new  construction. 
Construction  is  mainly  the  task  of  green  plants,  enabled  by  the  chloro- 
phyl  to  use  the  energy  of  sunlight  in  building  up  organic  substances 
from  minerals,  water  and  carbon  dioxide.  Destruction  is  caused 
mainly  by  animals  and  other  organisms  which  have  to  break  down 
organic  matter  in  order  to  exist.  These  two  factors  keep  the  atoms  of 
the  organic  world  in  perpetual  rotation. 

In  this  circulation  of  the  elements  it  is  necessary  that  all  compounds 
of  organic  nature  be  decomposed  finally  to  a  form  available  for  plant 
food..  If  this  were  not  the  case,  the  indestructible  compound  would 
sooner  or  later  accumulate  in  such  enormous  quantities  that  the 
elements  constituting  this  body  would  be  removed  entirely  from 
general  circulation.  Let  us  suppose,  as  an  illustration,  that  for  some 
unknown  reason,  all  urea  bacteria  on  earth  would  die.  Urea  could  be 
decomposed  no  more,  and  the  plants,  unable  to  use  urea  as  a  source  of 
nitrogen  in  place  of  nitrates,  would  get  but  little  benefit  out  of  stable 
manure.  All  urea  would  pass  gradually  undecomposed  into  rivers, 
lakes,  and  finally  into  the  ocean  where  it  would  accumulate  con- 
tinuously. The  enormous  quantities  of  nitrogen  taken  out  of  cir- 
culation would  cause  a  decreasing  growth  of  plants,  and  life  would 
soon  cease  because  of  lack  of  nitrogen.  For  this  reason  all  products  of 
living  organisms  must  be  further  broken  up  by  some  other  organisms, 

*  Prepared  by  Otto  Rahn. 

258 


NUTRITION   OF   MICROORGANISMS 


259 


and  we  find  that  the  destructive  work  is  to  a  large  extent  the  task  of 
microorganisms.  Many  products  of  organic  life  cannot  be  broken 
down  by  organisms  other  than  bacteria,  and  therefore  bacteria  are 
absolutely  necessary  for  the  circulation  of  the  elements  and  for  life  on 
earth.  Bacteria  and  green  plants  are  an  absolute  necessity  for  the 
maintenance  of  life,  the  one  breaking  down,  the  other  building  up, 
one  dependent  upon  the  products  of  the  other;  animals,  however,  could 
be  excluded  from  the  circle  without  interfering  with  a  continuation  of 
life  on  earth. 


nisms 


CartoAuclrafes 
fat,  Profein 

FIG.  114. — Carbon  cycle. 

CARBON  CYCLE. — Carbon  is  the  main  element  in  organic  nature,  and 
the  study  of  its  cycle  might  be  begun  with  its  simplest  compound, 
the  carbon  dioxide  of  the  air.  It  is  absorbed  in  this  condition  by  the 
green  plants,  and  is  changed  by  the  chlorophyl  granules  of  the  leaves  to 
organic  compounds  of  various  types,  either  to  carbohydrates  (cellulose, 
starch,  sugars)  or  to  fats,  or  to  protein  substances,  occasionally  to 
organic  acids  or  other  compounds.  The  plants  will  either  die  and  decay, 
or  will  be  eaten  by  animals.  In  the  first  case,  the  decay  will  be  caused 
exclusively  by  microorganisms;  if  the  plants  are  eaten,  they  will  be 
digested;  part  may  be  used  to  build  up  the  animal  body  or  stored  as 


260 


NUTRITION   AND   METABOLISM 


reserve  substances,  largely  fat  and  protein.  If  the  animal  dies,  a 
decomposition  process  will  take  place,  which  breaks  down  the  organic 
compounds  to  simpler  products  and  finally  the  carbon  will  be  com- 
pletely oxidized  to  carbon  dioxide.  Even  the  marsh  gas  which  might 
be  liberated  in  this  process  will  find  organisms  that  oxidize  it  to  carbon 
dioxide  and  water.  Every  product  will  find  an  organism  to  break  it 
up  further  until  it  is  completely  disorganized  and  the  carbon  atoms  can 
start  the  same  circulation  anew.  Undoubtedly  as  long  as  organic 
life  has  existed  on  earth,  microorganisms  have  been  present,  in  order 
to  render  the  dead  organic  matter  again  available  for  plant  and  animal 
life.  Fig.  114  gives  a  schematic  illustration  of  the  carbon  cycle;  the 
microbial  activity  is  marked  by  heavy  lines. 


Mtrates 


FIG.  115. — Nitrogen  cycle. 

NITROGEN  CYCLE. — Nitrogen  shows  the  same  continuous  change 
as  carbon.  Plants  take  up  nitrogen  in  mineral  form  usually  as  nitrates. 
The  plants  change  this  mineral  nitrogen  to  the  most  complex  bodies, 
proteins,  where  it  is  combined  with  the  other  elements  of  organic  nature. 
The  plants  may  be  eaten  by  animals;  part  of  the  protein  is  then  digested 
to  urea  or  hippuric  or  uric  acid,  which  in  turn  are  readily  decomposed 
by  microorganisms  to  ammonia  (Fig.  115).  Part  of  the  protein  will  be 
stored  in  the  growing  animals,  and  if  the  animal  dies,  the  body  will 
decay  or  putrefy,  and  the  nitrogenous  compounds  of  that  body  will 
pass  through  the  various  stages  of  decomposition  to  the  final  product, 


NUTRITION   OF   MICROORGANISMS  261 

ammonia.     Ammonia  is  then  oxidized  to  nitrites  and  nitrates,  when 
the  nitrogen  cycle  is  completed. 

There  is,  however,  one  discrepancy  in  this  cycle.  It  has  been 
mentioned  already  that  some  organisms  are  able  to  reduce  nitrates  to 
nitrogen  gas.  This  is  one  of  the  "leaks"  in  the  rotation  of  elements 
which  would  be  disastrous  to  organic  life  on  earth  if  there  were  no  means 
to  compensate  for  the  loss  of  nitrogen  in  circulation.  Imagine  what 
would  happen  if  there  were  no  such  compensation.  Part  of  the  nitrate 
in  the  soil  is  destroyed,  the  nitrogen  gas  escapes  into  the  air  and  is  as 
indifferent  as  the  nitrogen  of  the  atmosphere  lost  to  organic  life  forever. 


i/yclro* 


FIG.  116. — Sulphur  cycle. 

• 

More  nitrates  would  be  produced  from  decaying  organic  matter  and 
would  eventually  be  destroyed.  After  a  certain  time,  this  continuous 
loss  of  nitrogen  would  become  quite  noticeable  in  the  growth  of  plants; 
there  would  be  a  scarcity  of  nitrogen  in  soil,  since  part  of  it  is  lost  continu- 
ously. Finally,  the  plants  would  cease  to  grow  because  the  nitrogen  in 
the  soil  would  be  exhausted. 

The  compensation  for  this  destruction  of  available  nitrogen  is  found 
in  the  nitrogen-fixing  bacteria,  which,  either  living  in  symbiosis  with 
leguminous  plants  or  growing  independently  in  the  soil,  have  the  power 
to  use  the  atmospheric  nitrogen  for  the  formation  of  their  own  proto- 


262  NUTRITION  AND  METABOLISM 

plasm.  Thus,  organic  nitrogen  is  produced  from  nitrogen  gas  and  the 
continuance  of  organic  life  is  guaranteed. 

SULPHUR  CYCLE.— Little  more  can  be  said  about  sulphur,  since  the 
rotation  is  quite  similar  to  that  of  nitrogen.  Plants  will  take  sulphur 
usually  in  the  form  of  sulphates  and  make  protein  compounds  contain- 
ing a  certain  amount  of  sulphur  (Fig.  116).  These  bodies  are  either 
digested  by  higher  animals  or  broken  down  by  putrefaction  to  the 
final  product,  hydrogen  sulphide,  which  is  oxidized  by  the  sulphur 
bacteria  first  to  sulphur,  then  later  to  sulphates. 

PHOSPHORUS  CYCLE. — The  cycle  of  phosphorus  has  not  been  worked 
out  completely,  but  from  the  discussion  in  the  last  pages,  it  is  plainly 
seen  that  a  simple  cycle  very  much  like  the  ones  above  must  exist.  It 
is  probably  much  simpler  because  phosphorus  does  not  enter  as  easily 
into  organic  compounds  as  nitrogen. 


DIVISION  III 
PHYSICAL  INFLUENCES 


CHAPTER  I 
WATER  AS  A  PHYSICAL  FACTOR* 

It  has  been  indicated  already  that  water  has  the  capacity  as  a 
solvent  way  beyond  -any  other  substance;  it  has  a  function,  closely 
associated  with  its  solvent  powers  as  a  carrier,  in  which  solution  and 
mechanical  mixture  are  equally  important;  it  possesses  the  property 
of  diffusion,  which  enables  its  solutions  to  extend  where  other  solvents 
find  no  entrance;  it  possesses  much  surface  energy,  having  a  very  high 
rating;  and  it  fosters  ionization,  the  full  value  of  which  in  life's  reactions 
is  not  known.  Living  cells  have  been  shown  to  consist  of  a  high  per- 
centage of  water.  It  appears  as  if  water  were  the  body-medium  for  all 
physiological  reactions.  [See  pp.  187] 

OSMOTIC  PRESSURE. — In  the  organic  world  we  find  very  commonly 
membranes  which  will  allow  water  to  pass  through  but  retain  some 
compounds  dissolved  in  the  water.  Such  so-called  semi-permeable 
membranes  are  found  surrounding  the  protoplasm  of  cells.  They  are 
not  the  cell  wall,  but  separate  the  protoplasm  from  the  cell  wall. 
Similar  properties  are  found  in  parchment  paper,  pig's  bladder,  and 
other  organic  membranes. 

If  a  salt  solution  is  poured  in  water,  the  two  liquids  will  mix  in  a 
short  time  and  soon  every  smallest  portion  of  the  mixture  will  have  the 
same  concentration.  If  a  salt  solution  and  water  are  separated  by  a 
membrane  which  does  not  allow  the  salt  to  pass,  the  water  will  go 
through  the  membrane  toward  the  salt  with  a  certain  amount  of 
pressure.  This  pressure  depends  upon  the  nature  of  the  dissolved 
substance  as  well  as  upon  its  concentration. 

The  pressure  increases  in  direct  ratio  with  the  number  of  molecules 
in  solution.  Therefore,  a  compound  with  large  molecules  (cane  sugar) 

*  Prepared  by  Otto  Rahn. 

263 


264  PHYSICAL  INFLUENCES 

will  produce  a  lower  osmotic  pressure  than  one  with  small  molecular 
weight  (glycerin)  if  we  compare  solutions  of  equal  concentration. 
The  osmotic  pressure  of  protein,  starch  and  peptone  solutions  can  be 
measured  only  with  the  finest  instruments,  while  the  pressure  of  a  30 
per  cent  dextrose  solution  is  22  atmospheres.*  [See  pp.  173-177.] 

PLASMOLYSIS. — If  a  cell  is  brought  into  a  strong  solution  of  a  sub- 
stance which  cannot  pass  the  plasma-membrane,  this  substance  will 
cause  an  osmotic  pressure  and  the  concentration  in  the  cell  being  lower 
than  in  the  medium,  the  water  will  pass  out  from  the  cell  until  the  pres- 
sure inside  and  outside  is  the  same.  This  causes  a  shrinking  of  the 
protoplasm,  while  the  rigid  cell  wall  keeps  its  shape.  Such  plasmolyzed 
organisms  are  illustrated  in  Fig.  69,  page  89. 

While  plasmolysis  is  easily  demonstrated  with  the  cells  of  higher 
plants,  microorganisms  do  not  show  it  so  readily.  In  fact,  many  bac- 
teria, like  B.  subtilis,  Bact.  anthracis,  cannot  be  plasmolyzed  by  any 
concentration  of  salt  in  solution.  Others,  as  B.  coli,  B.  fluorescent, 
react  promptly.  But  even  though  many  are  killed,  the  rest  recover 
from  plasmolysis  after  a  few  hours,  and  appear  normal.  This  indicates 
that  the  salt  passes  slowly  through  the  plasma-membrane  and  thus 
increases  the  pressure  inside  the  cell  until  finally  the  inside  and  outside 
pressure  are  the  same  again. 

The  fact  that  many  microorganisms  show  no  plasmolysis  whatever 
is  explained  in  the  same  way.  These  organisms  probably  have  plasma- 
membranes  so  constructed  that  the  salts  diffuse  through  nearly  as  fast  as 
the  water.  An  absolute  exclusion  of  all  soluble  substances  by  the  mem- 
brane is  impossible  since  the  food  can  get  into  the  cell  only  by  diffusion 
through  the  membrane. 

The  resistance  of  various  microorganisms  against  concentrated 
solutions  depends  upon  the  organism  as  well  as  upon  the  dissolved  sub- 
stance. The  sodium  and  potassium  salts  of  the  common  mineral  acids 
act  upon  a  culture  nearly  in  proportion  to  their  osmotic  pressure,  but 
the  potassium  salts  always  retard  growth  a  little  less  than  the  sodium 
salts.  The  effect  of  salts  upon  microorganisms  is  therefore  not  due  to 
the  osmotic  pressure  only;  the  chemical  constitution  of  the  salts  also 
plays  an  important  rdle. 

The  different  functions  of  life  are  influenced  in  different  degrees  by 
concentrated  solutions.  Some  bacteria  will  multiply  but  not  form 

*One  atmosphere  equals  the  pressure  of  i  kg.  per  square  centimeter  or  about  15  pounds 
per  square  inch. 


WATER  AS   A   PHYSICAL  FACTOR  265 

spores  in  salt  solutions.  Molds  will  sometimes  show  a  good  growth  in 
concentrated  sugar  solutions  but  fail  to  produce  spores.  Bact.  anthracis 
loses  its  virulence  in  sea  water.  Often,  the  form  of  microorganisms  is 
affected  by  concentrated  solutions.  Some  bacteria  grow  more  spherical, 
others  become  elongated  or  distorted.  The  deforming  influence  is  not 
due  to  the  osmotic  pressure  only,  but  depends  mainly  upon  the  chemical 
character  of  the  salt;  magnesium  salts  especially  have  a  tendency  to 
produce  such  involution  forms. 

Salt  and  Sugar  Solutions. — Most  experiments  on  the  influence  of 
concentrated  solutions  have  been  carried  on  with  sodium  chloride,  be- 
cause of  its  wide  application  in  the  preservation  of  foods.  Most  micro- 
organisms, especially  the  rod-shaped  bacteria,  are  suppressed  by  a  salt 
concentration  of  8  to  10  per  cent.  At  15  per  cent  only  few  cocci  develop 
slowly,  while  some  species  of  Torula  grow  without  a  very  noticeable  re- 
tardation. Above  20  per  cent  the  Torula  are  practically  the  only 
organisms  which  can  develop.  They  are,  therefore,  found  in  all  food 
products  which  are  preserved  by  salt,  as  salted  pork,  beef,  fish,  butter, 
and  pickles,  often  in  nearly  a  pure  culture.  It  seems  that  they  are 
easily  overpowered  by  other  organisms  in  the  absence  of  salt,  but  in 
salted  food,  this  competition  is  eliminated. 

The  selective  influerfce  of  salt  is  used  in  some  fermented  products  to 
prevent  undesirable  fermentations.  This  is  true  in  sauerkraut  and 
brine  pickles,  where  the  desirable  bacteria  can  grow  in  the  presence 
of  salt  while  the  undesirable  ones  are  kept  away.  Possibly  the  salting 
of  butter  has  the  same  effects. 

Another  compound  of  great  practical  importance  is  cane  sugar,' 
which  is  the  standard  preservative  for  fruits  and  condensed  milk.  Its 
action  has  been  studied  mainly  upon  molds.  Theoretically,  dextrose 
should  be  expected  to  have  twice  as  strong  a  preserving  action  as  saccha- 
rose because  it  has  only  half  the  molecular  weight  and  consequently 
produces  twice  as  strong  an  osmotic  pressure  in  the  same  percentage  of 
concentration.  Its  preserving  effect  is  indeed  a  little  higher  than 
that  of  saccharose,  but  the  proportion  is  not  nearly  1:2.  The  common 
molds  are  extremely  resistant  to  strong  sugar  solutions,  about  60  to  70 
per  cent  of  cane  sugar  seems  to  be  the  limit  of  growth  for  Pemcillium 
and  Aspergillus  species.  Yeasts  can  also  grow  and  ferment  in  very  con- 
centrated solutions  while  bacteria  in  general  do  not  tolerate  solutions 
higher  than  15  to  40  per  cent,  though  many  exceptions  are  known. 


266  PHYSICAL   INFLUENCES 

Colloidal  Solutions. — In  order  to  determine  the  amount  of  water 
which  is  absolutely  necessary  for  microbial  proliferation,  only  such 
media  can  be  used  which  do  not  cause  osmotic  pressure.  If  B.  prodigio- 
sus  does  not  develop  in  a  10  per  cent  salt  solution,  this  is  not  due  to  lack 
of  moisture,  because  the  same  bacillus  will  grow  in  a  30  per  cent  sugar 
solution  which  contains  20  per  cent  less  moisture.  Another  factor  be- 
sides the  water  content  enters,  which  can  be  avoided  only  in  solutions 
without  osmotic  pressure. 

A  few  substances  are  known  to  give  such  solutions,  namely,  colloidal 
bodies  which  have  a  very  large  molecular  weight.  Their  osmotic  pres- 
sure even  in  very  concentrated  solutions  would  not  be  high  enough 
to  interfere  with  microbial  growth.  Among  these  colloidal  bodies 
are  found  egg  albumin,  gelatin,  peptones,  all  protein  substances; 
also  starch,  dextrin  and  gum  arabic  among  the  carbohydrates.  None 
of  these  substances  has  a  retarding  influence  upon  bacteria;  some  of 
them  can  be  mixed  with  water  in  all  proportions;  consequently,  they 
are  the  ideal  medium  to  test  the  water  requirements  of  microorganisms. 

Experiments  carried  on  with  gelatin,  powdered  meat,  crackers, 
bread  and  potato,  vary  but  little  in  results.  A  few  bacteria  cannot 
grow  in  a  medium  with  only  60  per  cent  water,  but  most  organisms 
develop  slowly  even  with  50  per  cent  water  arfd  some  may  be  able  to 
develop  with  only  40  per  cent.  Molds  can  grow  very  scantily  in  even 
more  concentrated  media.  Protozoa  probably  have  to  have  a  more 
diluted  medium  for  their  development  though  no  experiments  bearing 
upon  their  water  requirements  are  known  to  the  author. 

The  fact  that  in  a  colloidal  solution  growth  will  cease  if  the  moisture 
is  below  30  to  40  per  cent  does  not  necessarily  indicate  the  conclusion 
that  any  substance  with  less  than  30  per  cent  water  cannot  be  decom- 
posed. The  above  statement  refers  only  to  solutions,  while  in  natural 
media  as  dried  foods  or  soil,  a  combination  of  solid  and  dissolved 
substances  is  involved.  Butter  is  an  excellent  medium  for  many  bac- 
teria, yeasts,  and  molds,  though  it  contains  only  12  to  15  per  cent  of 
moisture.  If  butter  fat  were  soluble  in  water,  the  concentration  of  85 
parts  of  solid  in  15  parts  of  liquid  would  certainly  prevent  any  growth 
whatever,  but  fat  is  insoluble,  and  the  fat  particles  do  not  interfere 
at  all  with  the  growth  of  microorganisms  in  the  droplets  of  buttermilk 
distributed  all  through  the  butter.  The  concentration  in  these  small 
droplets  is  the  deciding  factor.  If  the  growth  of  microorganisms  in 


WATER   AS   A   PHYSICAL   FACTOR  267 

butter  is  to  be  prevented  by  salt,  it  is  unnecessary  to  give  any  attention 
to  the  fat;  the  bacteria  live  only  in  the  water  and  not  in  the  fat  globules. 
In  adding  3  per  cent  of  salt  to  a  butter  with  15  per  cent  of  moisture,  a 
brine  of  3  parts  of  salt  in  15  parts  of  water  is  produced;  in  other  words^ 
a  20  per  cent  brine,  because  salt  does  not  dissolve  in  the  fat.  Similar 
considerations  will  come  up  in  the  preservation  of  fruit,  vegetables,  meat, 
milk,  and  other  food  substances  by  drying  or  condensation. 

DESICCATION. — Microorganisms  do  not  die  immediately  after  the 
removal  of  the  water,  and  they  do  not  die  all  at  once  after  a  given  time. 
Death  through  drying  is  a  slow  and  regular  process.  Paul  and  his 
associates  found  that  the  number  of  bacteria-  dying  in  the  unit  of 
time  is,  under  constant  conditions,  proportional  to  the  number  sur- 
viving. If  we  had  1,000,000  cells  per  gram  in  the  beginning,  and  the 
death  rate  were  90  per  cent  per  day,  there  would  be,  at  the  end  of  each 
day,  10  per  cent  of  the  original  number  surviving.  This  would  give  the 
following  numbers  for  one  week: 

Beginning  1,000,000  cells  per  gram. 

After  i  day  100,000  cells  per  gram. 

After  2  days  10,000  cells  per  gram. 

After  3  days  1,000  cells  per  gram. 

After  4  days  100  cells  per  gram. 

After  5  days  10  cells  per  gram. 

After  6  days  i    cell  per  gram. 

After  7  days  o.i  cell  per  gram. 

This  table  shows  graphically  the  mode  of  death  of  dried  bacteria.  The 
number  of  cells  approaches  zero  without  ever  (at  least  theoretically) 
reaching  it.  From  one  cell  per  gram  after  six  days  we  do  not  come  to 
o  on  the  seventh,  but  to  one  cell  in  10  g.  and  on  the  eighth  day  one 
cell  in  100  g.  The  total  number  dying  in  the  first  day  is  much  larger 
than  that  dying  on  the  sixth  day,  but  the  rate  is  constant,  90  per  cent 
of  the  number  surviving.  This  regularity  has  been  found  with  bacteria 
dying  from  various  causes,  and  it  is  commonly  compared  with  the 
simplest  chemical  processes,  the  monomolecular  reactions. 

Paul  and  his  associates  found  further,  that  the  death  through  drying 
is  caused  by  an  oxidation  process;  in  pure  oxygen  bacteria  died  much 
faster.  The  poisonous  effect  of  oxygen  upon  moist  bacteria  has  already 
been  pointed  out  on  page  228. 

Most  resistant  to  drying  are  the  spores  of  bacteria;  mold  spores, 


268  PHYSICAL   INFLUENCES 

too,  show  considerable  resistance,  while  some  bacteria,  e.g^B.carotarum 
and  Ps.  radicicola,  are  readily  killed. 

The  resistance  of  microorganisms  is  influenced  greatly  by  the  me- 
'dium  on  which  they  are  placed  for  drying.  Hansen  found  that  yeast 
cells  dried  on  cotton  were  still  alive  after  two  to  three  years,  while  if 
dried  on  platinum  wire  some  died  in  five  days  and  others  lived  as  long  as 
100  days.  Compressed  beer-yeast  mixed  and  dried  with  powdered  char- 
coal kept  as  long  as  ten  years;  Ps.  radicicola  dried  on  a  cover-glass 
or  filter  paper  died  within  twenty-four  hours;  on  seeds,  this  same  organism 
was  still  alive  after  fourteen  days  and  in  the  dried  nodules  of  legumes  a 
few  cells  were  able  to  reproduce  after  more  than  two  years.  Soil  con- 
taining an  average  number  of  17,000,000  bacteria  per  gram  was  dried  for 
two  years;  the  total  number  of  organisms  averaged  then  3,250,000,  20 
per  cent  of  the  bacteria,  therefore,  could  resist  desiccation.  Dried  cul- 
tures of  microorganisms  are  commonly  sold  for  several  purposes,  as 
dairy-starters  and  the  so-called  "magic  yeast"  and  "yeast  foam"  used  for 
bread-making.  Such  cultures  are  dried  on  milk,  sugar,  starch,  flour  or 
similar  porous  and  absorbing  material.  Starters  are  usually  guaranteed 
only  for  a  certain  length  of  time,  from  one  to  twelve  months.  The 
advantage  of  the  dry  culture  is  its  better  keeping  qualities.  Liquid 
cultures  produce  substances  harmful  to  themselves,  and  die  rapidly 
after  a  short  time,  while  the  dry  cultures  show  little  change. 

The  resistance  of  pathogenic  bacteria  to  desiccation  is  of  consider- 
able importance  in  the  spreading  of  contagious  diseases.  Many  patho- 
genic bacteria  die  after  desiccation  of  a  few  hours  to  a  few  days,  and 
spreading  of  such  diseases  by  dust  is  highly  improbable.  Protozoa  of 
soil  decrease  in  number  by  drying,  but  all  are  not  killed. 


CHAPTER  II 
INFLUENCE  OF  TEMPERATURE* 

Temperature,  as  well  as  moisture,  is  one  of  the  most  important  fac- 
tors of  life.  It  is  so  important  that  the  most  highly  developed  animals 
protect  themselves  by  a  very  complicated  mechanism  of  regulation 
against  changes  of  temperature;  the  life  processes  of  such  animals  will 
take  place  at  a  temperature  nearly  constant  from  birth  to  death.  This 
causes  the  metabolism  of  warm-blooded  animals  to  be  different  from 
that  of  all  other  organisms.  The  metabolism  of  the  warm-blooded 
animals  takes  place  at  a  constant  temperature.  The  required  amount 
of  food  is  constant  except  for  the  part  that  is  used  for  heating  the  body; 
at  lower  temperatures,  more  heat-producing  material  is  used  and  the 
result  is  that  warm-blooded  animals  require  more  food  at  lower  tempera- 
ture. All  other  organisms,  reptiles  as  well  as  bacteria,  have  the  tem- 
perature of  their  environment  and  the  decrease  of  temperature  will 
decrease  the  intensity  of  metabolism  as  it  retards  any  other  chemical 
process.  The  lower  the  temperature,  the  less  food  is  required  by  all 
lower  organisms. 

There  are,  of  course,  limits  to  the  favorable  influence  of  high  tempera- 
tures. Growth  and  metabolism  of  microorganisms  will  increase  with 
rising  temperature  to  a  certain  point,  called  the  optimum  temperature, 
and  beyond  this  point  the  rate  of  growth  will  fall  off  rapidly  and  soon 
cease  entirely.  The  highest  temperature  at  which  growth  can  take 
place  is  called  the  maximum  temperature.  Correspondingly,  the  mini- 
mum temperature  of  an  organism  is  the  lowest  point  at  which  growth  can 
take  place. 

THE  OPTIMUM  TEMPERATURE  which  allows  the  fastest  growth  will  be 
quite  different  for  different  species.  Groups  of  bacteria  are  known 
which  develop  only  at  very  high  temperatures  and  others  for  which  room 
temperature  is  too  high.  The  temperature  requirement  is  largely  de- 
pendent upon  the  natural  habitat  of  the  organisms.  The  bacteria  of 

•  Prepared  by  Otto  Rahn. 

269 


270 


PHYSICAL   INFLUENCES 


the  polar  sea  and  of  a  lagoon  near  the  equator  will  very  probably 
have  different  optimum  temperatures  because  of  the  acclimatization 
and  selection  which  has  been  taking  place  for  centuries. 

The  great  majority  of  bacteria  and  related  organisms,  in  fact  of  all 
living  organisms,  except  in  a  few  instances,  has  its  optimum  tem- 
perature between  20°  and  40°.  The  optimum  temperature  of  an 
organism  is  generally  somewhat  higher  than  the  average  temperature 
of  its  natural  habitat. 

The  following  table  shows  the  data  obtained  for  a  few  microor- 
ganisms. 


Temperatures 

Minimum 

Optimum 

Maximum 

Penicillium  glaucum              

1.5° 

2S°-270 

3i°-36° 

Aspcrzillus  niger 

7°-lO° 

M0-.^0 

40°-43° 

Saccharomyces  cerevisicz  I.        

i°-3° 

28°-30° 

40° 

Saccharomyces  pasteurianus  I 

o   <?° 

2S°-30° 

74° 

Bacterium  phosphor  cum       

below  o° 

i6°-i8° 

28° 

Bacillus  subtilis  

6° 

30° 

50° 

Bacterium  anthracis                    

10° 

30°—  37° 

43° 

Bacterium  ludwigii  

So° 

55°-57° 

80° 

THE  MINIMUM  TEMPERATURE  or  the  lowest  limit  of  growth  is  usually 
farther  from  the  optimum  than  the  maximum  temperature.  It  will 
vary  with  the  organisms  just  as  do  the  other  cardinal  points.  But 
there  is  a  natural  limit  drawn  by  the  freezing-point  of  the  nutrient 
liquid.  Not  all  organisms  can  grow  at  such  low  temperatures,  in  fact 
the  greater  number  does  not  develop  below  6°  to  10°.  Those  that  can 
grow  at  the  freezing-point  will  be  inhibited  by  the  solidification  of  the 
water  in  the  nutrient  medium,  for  if  the  water  is  frozen,  food  cannot 
diffuse  intb  the  cells  and  therefore,  all  life  processes  are  checked.  If 
freezing  is  prevented  by  adding  salts  or  other  soluble  substances  which 
lower  the  freezing-point,  growth  may  continue  even  below  o°.  Milk 
freezes  at  about  —  0.5°.  Bacteria  are  found  to  multiply  in  it  as  long  as 
it  is  not  entirely  solid.  A  certain  yeast  multiplied  slowly  in  salted  but- 
ter kept  at  about  —  6°. 


INFLUENCE    OF    TEMPERATURE  271 

x 

The  number  of  microorganisms  that  developed  at  the  freezing- 
point  was  found  to  be: 

In  i  c.c.  of  market  milk,  up  to    1,000  germs. 
In  i  c.c.  of  sewage,  up  to    2,000  germs. 

In  i  g.     of  garden  soil,     up  to  14,000  germs. 

THE  MAXIMUM  TEMPERATURE  is  usually  about  10°  to  15°  higher 
than  the  optimum.  The  development  of  microorganisms  above  the 
optimum  temperature  is  not  quite  normal;  there  is  a  great  tendency 
toward  involution  forms.  The  mycelium  of  molds  grown  near  the 
maximum  temperature  appears  unhealthy  and  pathogenic  bacteria 
lose  part  of  their  virulence.  This  loss  of  virulence  is  made  use  of  in 
the  preparation  of  attenuated  cultures  for  vaccines. 

The  maximum  temperature  varies  with  different  species  of  bac- 
teria. Most  bacteria  do  not  grow  above  45°,  but  with  some  of  them 
the  maximum  temperature  ,is  considerably  lower.  Bad.  phosphor eum 
dies  if  exposed  for  a  few  hours  at  30°;  others  may  require  still  lower 
temperatures.  The  average  organisms  found  in  water,  soil,  milk,  and 
the  body,  which  have  their  optimum  near  30°  to  38°,  do  not  grow  higher 
than  about  45°.  There  are  very  noticeable  exceptions  to  these,  such 
as  the  physiological  group  known  as  thermophilic  bacteria. 

These  extraordinary  organisms  have  their  maximum  between  70° 
and  80°,  a  temperature  which  coagulates  albumin.  Corresponding  to 
the  high  maximum  the  thermophiles  have  a  very  high  optimum,  and 
the  minimum  lies  with  most  of  these  species  above  30°.  These  or- 
ganisms are  found  in  soil,  sewage,  ensilage  and  occasionally  in  milk. 
They  find  the  temperature  suitable  for  their  life  only  under  extra- 
ordinary circumstances,  as  in  fermenting  manure  piles,  in  silos,  in 
self-heating  hay  and  similar  organic  material  that  develops  a  high 
temperature  by  fermentation.  Some  hot  springs  have  a  very  remark- 
able flora  of  thermophilic  bacteria. 

The  range  of  temperature  within  which  growth  is  possible,  is  very 
uniformly  35°  to  45°;  the  starting  points  and  end-points  of  this  range 
vary  greatly,  while  the  total  range  is  quite  constant,  except  for  some 
bacteria  adapted  to  special  conditions,  such  as  some  pathogenic  bac- 
teria. The  temperature  relations  of  bacteria  can  be  shown  graphically 
by  using  as  ordinate  the  rate  of  growth;  as  abscissa  the  temperature 


272  PHYSICAL  INFLUENCES 

V 

BIOLOGICAL  SIGNIFICANCE  OF  THE  CARDINAL  POINTS  OF  TEMPERA- 
TURE.— The  importance  of  the  temperature  requirements  of  certain 
organisms  to  the  r61e  they  play  in  nature  can  be  illustrated  by  a  few 
examples.  Most  molds  cannot  cause  disease  in  man  and  warm- 
blooded animals  because  their  maximum  temperature  is  below  the 
body  temperature.  Exceptions  are  some  Aspergilli  and  Mucorinece. 
Pathogenic  microorganisms  must  have  their  optimum  temperature 
coincide  with  that  of  their  host. 

Organic  substances  may  undergo  a  different  change  at  different 
temperatures.  The  biochemical  changes  in  soil  may  not  be  the  same 
in  northern  Canada  and  near  the  Gulf  of  Mexico.  Even  the  warm  and 
cold  season  of  the  same  climate  is  apt  to  change  not  only  the  rate  of 
decomposition  but  possibly  the  products.  Perhaps  the  most  striking 
example  in  this  respect  is  the  decomposition  of  ordinary  market  milk 
kept  at  different  temperatures.  Such  milk  contains  a  great  variety 
of  microorganisms;  at  various  temperatures  different  types  will  pre- 
dominate, while  the  remainder  are  retarded  or  inhibited  by  unfavor- 
able temperature  conditions  and  by  the  products  of  the  dominant  type 
of  bacteria.  If  milk  is  kept  at  about  the  freezing-point,  only  a  few 
organisms  will  develop  slowly,  but  after  a  certain  time  their  number 
will  increase  to  many  million  cells  per  c.c.  There  is,  however,  no  appar- 
ent change;  no  acid  or  deterioration  can  be  discovered  by  the  taste 
though  chemical  analysis  proves  the  presence  of  hydrogen  sulphide 
and  ammonia.  Between  15°  and  25°,  milk  will  sour  in  about  thirty- 
six  to  forty-eight  hours,  giving  a  firm  curd  of  an  agreeable  flavor 
without  whey  or  gas;  later  Oidium  lactis  destroying  the  acid  develops 
on  the  surface.  Near  body  temperature  the  milk  will  lopper  in  twenty- 
four  hours,  the  curd  is  usually  contracted,  a  large  quantity  of  whey 
is  extruded,  and  much  gas  is  produced  by  Bact.  aero  genes  and  B.  coli. 
The  odor  is  disagreeable  and  later  butyric  acid  is  produced;  eventu- 
ally the  lactic  acid  increases  further  by  the  action  of  Bact.  bulgaricum. 
If  kept  above  50°  the  milk  either  keeps  permanently,  or  a  decomposi- 
tion by  thermophilic  bacteria  begins  which  is  either  an  acid  fermenta- 
tion followed  by  digestion  or  a  complete  putrefaction,  depending  upon 
the  species  of  thermophilic  organism  that  happens  to  be  in  the  milk 
sample.  Thus  there  can  be  induced  in  the  same  substance,  contain- 
ing the  same  organisms  at  the  start,  four  entirely  different  types 
of  decomposition  merely  by  the  difference  of  temperature. 


INFLUENCE    OF   TEMPERATURE  273 

This  indicates  the  importance  of  temperature  regulation  in  the  fer- 
mentation industries.  Even  pure  cultures  may  give  different  products 
if  working  at  different  temperatures.  Cream  ripened  with  a  pure 
culture  starter  at  too  high  a  temperature  will  have  a  sharp  acid  flavor. 
The  cold  curing  of  cheese  has  become  a  very  common  practice  because 
of  the  much  improved  flavor.  Bioletti  claims  that  the  value  of  the  dry 
California  wines  would  be  doubled  if  the  fermentation  were  carried 
on  generally  at  a  lower  temperature. 

END-POINT  OF  FERMENTATION. — Another  question  is  the  relation 
between  the  end-point  of  fermentation  and  the  temperature.  Of  the 
few  data  existing,  many  indicate  that  at  a  lower  temperature  the  final 
fermentation  goes  farther  than  at  a  higher  temperature.  Muller- 
Thurgau  found  that  under  exactly  the  same  conditions  with  the  tem- 
perature as  the  only  varying  factor  the  following  final  amounts  of 
alcohol  were  produced  by  a  pure  culture  of  yeast: 

At  36° 3 .8  per  cent  alcohol. 

At  27° 7.5  per  cent  alcohol. 

At  18° 8.8  per  cent  alcohol. 

At    9° 9.5  per  cent  alcohol. 

Concerning  the  lactic  fermentation  some  investigators  find  no  differ- 
ence in  the  end-point,  while  others  obtained  results  similar  to  the  re- 
sults with  alcohol.  With  three  strains  of  Bact.  lactis  acidi  were  ob- 
tained after  thirty-four  days,  by  C.  W.  Brown: 

A  B  C 

At  37°   0.89  per  cent  0.87  per  cent  0.60  per  cent  of  lactic  acid. 

At  30°    i. oo  per  cent  0.96  per  cent  o. 8 1  per  cent  of  lactic  acid. 

At  18°    i. 08  per  cent  1.06  per  cent  0.88  per  cent  of  lactic  acid. 

At    6°   0.70  per  cent  0.73  per  cent  0.62  per  cent  of  lactic  acid. 

These  results  are  quite  logical  and  perhaps  can  be  explained  by 
the  recognized  experience  that  all  products  of  fermentation  tend  to 
check  the  process  of  fermentation,  and  that  any  chemical  product 
or  substance  acts  the  more  vigorously  upon  any  life  process  the  higher 
the  temperature.  The  same  amount  of  alcohol  that  will  still  allow  a 
slow  fermentation  at  10°  may  check  the  fermentation  entirely  at  20°. 
Naturally  the  rate  of  fermentation  in  the  beginning  will  be  higher  at 
the  higher  temperature  but  the  end-point  is  lower.  The  end-point  of 
the  lactic  cultures  A,  B,  and  C  at  6°  is  probably  not  final,  because 

18 


274  PHYSICAL   INFLUENCES 

thirty-four  days  is  a  short  time  of  growth  at  so  low  a  temperature. 
Above  the  optimum,  the  rate  of  decomposition  will  decrease  rapidly 
with  the  rising  temperature  and  the  end-point  will  also  be  lower. 

FREEZING. — The  discussion  of  the  relation  of  temperature  to 
microorganisms  has  so  far  considered  only  the  temperatures  within 
the  limits  of  growth.  However,  the  temperatures  below  the  minimum 
and  above  the  maximum  are  also  of  greatest  importance.  If  bacteria 
are  cooled  below  their  minimum  temperature  they  do  not  die  immedi- 
ately. They  remain  alive  in  a  dormant  condition  ready  to  multiply 
as  soon  as  the  temperature  rises.  Even  the  freezing  of  a  liquid  will 
not  kill  them  immediately.  Of  course,  they  cannot  multiply  in  ice, 
because  they  have  no  water,  consequently  no  food,  and  they  cannot 
thaw  the  ice  to  get  their  water  and  food  for  lack  of  body  temperature 
of  their  own.  As  long  as  liquids  are  frozen  solid  the  bacteria  in  them 
will  remain  dormant  much  like  dried  organisms,  and  like  them  their 
number  will  decrease  very  slowly.  An  example  is  given  in  the  follow- 
ing table  relevant  to  the  number  of  bacteria  in  frozen  milk  (after 
Bischoff).  The  decrease  in  numbers  is  not  very  uniform,  since  there  are 
many  different  bacteria  in  milk,  but  the  general  tendency  is  the  same 
as  in  the  dried  bacteria. 

Milk  kept  at  3°  to  -7° 

Freshly  frozen , 200,000  bacteria  per  c.c. 

After    i  day 105,500  bacteria  per  c.c. 

After    2  days 72,300  bacteria  per  c.c. 

After    3  days 62,000  bacteria  per  c.c. 

After   4  days 46,400  bacteria  per  c.c. 

After    7  days 44,000  bacteria  per  c.c. 

After  14  days 40,500  bacteria  per  c.c. 

After  21  days 30,300  bacteria  per  c.c. 

After  35  days 22,500  bacteria  per  c.c. 

After  49  days 14,200  bacteria  per  c.c. 

The  table  shows  plainly  that  it  is  impossible  to  sterilize  milk  by 
freezing,  but  as  long  as  it  is  frozen  it  will  keep;  there  is  no  possibility 
of  any  microorganisms  decomposing  a  frozen  liquid,  for  the  organisms 
need  water  above  all.  If  food  substances  change  in  cold  storage 
(and  some  food  products  do  deteriorate),  this  must  either  be  due  to 
changes  other  than  microbial  or  the  material  was  not  completely 
frozen  as  is  probably  the  case  with  salted  butter. 


INFLUENCE    OF    TEMPERATURE  275 

After  bacteria  are  once  frozen,  they  do  not  seem  to  be  affected  by 
any  lower  temperature.  Macfadyen  and  Rowland  found  that  they 
tolerate  very  low  temperatures  remarkably  well.  Many  bacteria 
were  not  killed  by  a  twenty  hours'  exposure  to  the  temperature  of 
liquid  hydrogen  (  —  252°).  Yeasts  are  not  quite  so  resistant  and  the 
mycelium  of  most  molds  is  easily  destroyed  by  freezing,  while  the  spores 
are  hardier. 

THERMAL  DEATH-POINT. — Heating  above  the  maximum  tempera- 
ture is  quite  harmful  to  bacteria,  and  the  amount  of  injury  increases 
with  the  temperature.  Recent  experiments  have  shown  that  heat  does 
not  kill  bacteria  instantaneously,  but  that  we  have  an  orderly  process 
as  in  the  case  of  death  by  drying.  This  can  be  observed  only  in  a 
very  narrow  range  of  temperature,  however,  since  the  death  rate  rises 
very  rapidly  with  the  increase  of  temperature.  10°  increase  may  make 
the  death  rate  ten  to  one  hundred  times  as  great,  and  death  is  almost 
instantaneous.  For  most  practical  purposes,  it  is  sufficient  to  state 
the  time  and  temperature  necessary  to  bring  about  complete  sterili- 
zation. It  has  become  customary  to  define,  as  the  thermal  death- 
point,  the  lowest  temperature  at  which  a  culture  will  be  killed  in  ten 
minutes.  As  most  bacteriologists  will  use  very  nearly  the  sametech- 
nic,  they  will  have  fairly  uniform  numbers  of  cells  to  start  with, 
and  therefore  obtain  fairly  uniform  results. 

The  thermal  death-point  does  not  depend  upon  the  species  and 
the  temperature  only.  It  varies  with  the  age  of  the  culture  since 
older  cells  are  less  resistant  than  younger  ones  especially  if  heated  in 
their  own  products.  The  medium  in  which  the  organisms  are  heated 
is  also  of  great  significance.  The  fact  that  acid  liquids,  as  fruit  juices, 
are  more  easily  sterilized  than  neutral  meat  or  vegetables  is  largely 
due  to  a  chemical  (poisonous)  action  of  the  acids  upon  the  bacteria. 
But  the  greater  resistance  of  tubercle  bacteria  in  the  sputum  compared 
with  those  suspended  in  salt  solution  cannot  be  so  readily 
accounted  for. 

A  necessary  factor  for  the  prompt  destruction  of  organisms  by 
heat  is  the  presence  of  moisture.  The  resistance  of  dry  organisms 
is  remarkably  higher  than  that  of  the  same  organisms  in  a  liquid  cul- 
ture. The  following  table  shows  the  death-point  of  yeast  cells  and 
spores  in  a  dry  and  moist  state. 


276  PHYSICAL  INFLUENCES 

THERMAL   DEATH-POINT  OF  DRY  AND   MOIST  YEAST 


Moist 

Dry 

Moist 

Dry 

Pale  ale  yeast 

6c° 

| 

QC°—  TO^° 

6c°-7o° 

TIC0-T2C° 

Hofbrau  yeast.     . 

rr° 

8<°-  00° 

6c° 

ilO       1*O 
TTC0-T  20° 

Saccharomyces  pasteurianus 

5o°-55° 

ioo°-io5° 

60°  • 

H5° 

RESISTANCE  or  SPORES. — The  organisms  most  resistant  to  heat  are 
the  spores  of  certain  bacteria.  In  the  chapter  on  moisture  require- 
ments attention  has  been  called  to  the  great  resistance  of  spores  to 
drying.  We  find  the  same  exceptional  resistance  to  high  temperatures. 
Boiling  heat  will  not  kill  spores  readily.  Some  bacterial  spores  can 
stand  the  temperature  of  100°  for  several  hours.  In  order  to  kill  spores 
in  one  heating  the  temperature  must  rise  to  about  110°  for  fifteen  to 
thirty  minutes;  this  can  be  accomplished  only  by  heating  under  pres- 
sure. This  is  not  always  advisable  for  sterilizing  food  substances. 
While  vegetables  are  usually  sterilized  under  pressure  without  losing 
much  of  their  palatability,  other  foods  like  milk  are  changed  materially 
in  taste  and  appearance.  To  prevent  these  changes,  discontinuous 
sterilization  is  sometimes  used.  This  is  based  upon  the  following 
principle. 

If  milk  or  any  other  medium  is  heated  to  100°  for  about  fifteen  min- 
utes, all  living  cells  of  bacteria,  yeasts  and  molds  will  be  killed  except  a 
few  spores  of  bacteria.  After  cooling,  these  spores  will  germinate  under 
suitable  conditions  and  the  vegetative  cells  thus  appearing  instead  of  the 
resistant  spores  are  easily  killed  in  a  second  heating.  A  third  heating 
is  necessary  in  order  to  kill  any  vegetative  cells  which  may  have  devel- 
oped from  spores  not  yet  germinated  before  the  second  heating.  It  is 
essential  to  have  the  time  between  two  heatings  long  enough  to  allow  the 
germination  of  spores,  and  not  too  long  to  permit  formation  of  new 
spores.  It  is  customary  to  heat  on  three  successive  days  for  fifteen 
minutes  each  time.  In  this  case,  sterilization  is  usually  complete, 
while  a  forty-five  minutes'  heating  at  once  is  not  sufficient  to  guarantee 
sterilization.  Among  the  substances  that  are  very  easily  sterilized  are 
cider  and  other  fruit  juices,  while  milk  and  soil  are  the  most  difficult 
materials  to  sterilize. 


INFLUENCE    OF    TEMPERATURE  277 

Dry  spores  will  resist  still  higher  temperatures  than  moist  spores. 
Some  dry  spores  survive  an  exposure  to  140°  or  150°  for  ten  minutes. 
It  requires  a  very  high  temperature  to  sterilize  glass,  cotton,  gauze,  and 
instruments  with  dry  heat.  A  discontinuous  sterilization  of  dry  mate- 
rial is  useless,  since  the  spores  will  not  germinate  without  moisture, 
therefore  their  resistance  remains  unaltered. 

The  spores  of  molds  are  more  resistant  than  the  mycelium,  but  if 
moist,  they  all  die  at  100°.  The  dry  mold  spores  can  tolerate  a  some- 
what higher  temperature,  but  not  as  high  as  the  spores  of  many  bacteria. 
Yeast  spores  and  yeast  cells  are  very  much  alike  in  their  resistance  to 
heat.  The  table  on  page  276  shows  hardly  any  difference  between  their 
resistance. 


CHAPTER  III 
INFLUENCE  OF  LIGHT  AND  OTHER  RAYS* 

Microorganisms  in  their  natural  environment  are  temporarily  but 
not  usually  exposed  to  light.  The  organisms  of  decay,  living  in  soil,  in 
foods,  in  the  intestines  of  animals,  will  only  occasionally  come  in  con- 
tact with  the  direct  rays  of  the  sun.  Water  bacteria  and  the  organisms 
on  the  surface  of  plants  and  animals  are  more  commonly  exposed  to  the 
sun. 


FIG.  117. — These  plates  were  heavily  inoculated  with  B.  coli  and  B.  prodigiosus 
respectively  and  then  were  exposed,  bottom  side  up,  to  the  direct  rays  of  the  sun, 
for  four  hours.  On  the  instant  of  exposure,  a  figure  O  cut  from  black  paper  was 
pasted  to  the  plate  shading  the  bacteria  underneath.  After  one,  two  and  three  hours 
the  corresponding  figures  were  pasted  to  the  plates.  The  above  picture  was  taken  2 A 
hours  after  exposure,  proving  that  three  or  four  hours  of  direct  sunlight  weaken  and 
and  may  even  kill  bacteria.  B.  prodigiosus  proved  more  sensitive  than  B.  coli. 
(Original.) 

The  influence  of  light  varies  with  its  intensity.  Direct  sunlight 
has  a  very  harmful  effect  upon  microorganisms.  Most  bacteria  are 
killed  by  direct  sunlight  in  a  few  hours;  the  time  depends  upon  the 
organism  as  well  as  upon  the  intensity  of  light;  this  again  varies  with 

*  Prepared  by  Otto  Rahn. 

278 


INFLUENCE    OF   LIGHT  AND    OTHER   RAYS 


279 


the  amount  of  moisture  and  dust  in  the  atmosphere,  with  the  time  of 
the  day  and  with  the  season;  an  absolute  measure  for  the  action  of  light 
cannot  be  fixed,  therefore,  as  easily  as  with  the  action  of  heat  in  the  ther- 
mal death-point.  The  different  colors  of  the  spectrum  do  not  act 
alike;  the  part  of  the  spectrum  from  red  to  green  is  practically  without 
influence  upon  microorganisms,  while  the  blue  light  acts  strongest 
and  the  intensity  decreases  in  the  violet  and  ultra-violet.  In  carrying 
on  experiments  with  the  influence  of  light,  it  must  be  remembered  that 
glass  absorbs  ultra-violet  rays,  and  further  that  the  heating  of  the 
medium  by  direct  radiation  must  be  avoided  (Fig.  117). 


FIG.  1 1 8. — Phototropism  of  Rhizopus  nigricans.     The  mold  is  grown  on  gelatin  with 
diffused  light  coming  from  right  side.     (Original.) 

Yeasts,  molds,  and  bacteria  and  probably  Protozoa  are  equally  sensi- 
tive to  light.  Even  the  spores  of  most  bacteria  do  not  show  a  greater 
resistance  to  light,  while  the  mold  spores  are  an  exception.  The  col- 
ored spores  of  the  Penicillium,  Aspergillus  and  Mucor  species  can  be 
exposed  to  light  for  a  long  time  without  being  killed,  but  the  colorless 
spores  of  Oidium  and  Chalara  show  no  increased  resistance.  It  is  sup- 
posed that  the  pigment  in  mold  spores  is  a  protection  against  light.  This 
is  not  true  with  the  pigment  of  bacteria.  The  colored  and  colorless 
strains  of  pigmented  bacteria  show  no  difference  in  their  resistance  to 
light.  The  only  exceptions  are  the  so-called  purple  bacteria.  These 
peculiar  organisms,  many  of  which  feed  on  hydrogen  sulphide,  seem  to 


280 


PHYSICAL  INFLUENCES 


thrive  better  in  light  than  without  it.  Direct  sunlight  does  not  kill 
them,  it  rather  attracts  them  and  they  move  toward  the  light.  This  is 
called  phototaxis  or  heliotaxis.  The  pigment,  bacteriopurpurin,  does 
not  take  the  place  of  chlorophyl,  however,  since  the  bacteria  do  not  pro- 
duce oxygen  in  light  and  always  need  organic  food. 

The  effect  of  light  upon  microorganisms  is  mainly  brought  about  by 
a  chemical  change  in  the  protoplasm,  and  also,  to  some  extent,  by  a 
chemical  change  in  the  medium,  namely  the  formation  of  a  peroxide  or  a 
similar  oxidizing  agent. 

The  germicidal  action  of  light  is  of  importance  in  the  purification  of 
rivers.  It  is  applied  also  in  curing  diseases  of  the  skin,  as  lupus  and 


FIG.  119. — Two  cultures  of  an  Aspergillus^one  grown  in  the  dark  the  other  in 
diffused  light,  showing  rings.     (Original.) 

leprosy,  by  exposing  the  diseased  parts  to  a  very  concentrated  light  of 
the  electric  arc.  This  light  contains  plenty  of  blue  and  violet  rays  and 
is  preferable  to  sunlight  because  it  is  always  ready  for  use  and  its  com- 
position and  intensity  can  be  controlled  easily.  Ultra-violet  light  is 
used  in  the  sterilization  of  water  and  of  milk. 

Diffuse  light  is  not  nearly  as  harmful  to  microorganisms  as  direct 
sunlight.  Long  exposures  to  diffuse  light  will  kill  most  bacteria,  while 
molds  are  not  at  all  sensitive.  They  rather  like  a  very  dim  light,  and 
many  molds  grown  in  a  dark  room  with  light  only  from  one  side  will 
grow  toward  the  light.  This  property,  which  is  characteristic  for  all 
green  plants,  is  called  heliotropism  or  phototropism  (Fig.  118).  It  has 


INFLUENCE    OF   LIGHT   AND    OTHER   RAYS  281 

been  found  that  molds  produce  mycelium  mostly  in  the  dark,  while  in 
daylight  sporangia  are  produced  mainly.  This  difference  in  the  devel- 
opment during  the  day  and  during  the  night  accounts  for  the  concentric 
rings  which  are  quite  commonly  found  in  older  mold  colonies,  and 
which  indicate  the  age  of  the  culture  (Fig.  119).  Similar  rings  are 
occasionally  found  with  yeast  and  bacterial  colonies,  and  are  possibly 
due  to  the  same  influence  of  light. 

X-RAYS. — Of  other  rays,  the  invisible  X-rays  and  the  radium  rays 
have  attracted  the  attention  of  bacteriologists  and  physiologists.  It 
is  known  that  the  X-rays  will  destroy  living  tissue  by  long  exposures; 
microorganisms  cannot  be  considered  less  resistant.  X-rays  are  used 
in  the  treatment  of  microbial  diseases  of  the  scalp  and  skin. 

RADIUM  RAYS  are  not  so  well  known,  and  their  bactericidal  action  is 
doubtful.  .The  treatment  of  certain  bacterial  diseases  has  been 
attempted,  but  it  has  not  been  applied  as  generally  as  yet  as  the  X-ray 
method.  The  sterilization  of  milk  and  possibly  other  foods  by  this 
method  has  been  suggested,  but  the  practical  application  is  at  present 
quite  improbable  because  of  the  cost  and  the  uncertainty  of  the  results. 


CHAPTER  IV 
INFLUENCE  OF  ELECTRICITY* 

The  influence  of  electricity  upon  microorganisms  is  much  less  than 
one  might  perhaps  expect,  if  the  electricity  as  such  is  considered.  A 
direct  electric  current  passing  through  a  nutrient  medium  will,  of  course, 
cause  electrolysis  which  is  usually  manifested  by  the  formation  of  acid 
on  the  positive  pole  and  of  alkali  on  the  negative  pole.  The  acid  and 
alkali  will  kill  microorganisms,  as  is  discussed  in  the  chapter  on  chemical 
influences.  In  this  case,  it  is  not  the  electricity  itself  that  destroys  the 
bacteria.  It  is  also  possible  to  kill  bacterial  cultures  by  passing  an 
alternating  current  through  the  medium  for  some  time.  No  electrolysis 
takes  place  in  this. case,  still  it  is  not  the  current  that  acts  directly  upon 
the  organisms,  but  rather  the  heat  produced  by  the  current  passing 
through  a  medium  of  high  resistance.  If  the  culture  is  cooled  properly 
the  influence  of  the  current  is  insignificant  if  at  all  noticeable.  When- 
ever electricity  is  applied  against  microorganisms  the  effect  is  con- 
sidered electrochemical. 

The  electrical  current  is  used  in  a  very  small  way  in  the  purification 
of  sewage.  The  sewage  passes  between  two  iron  plates  which  represent 
the  two  poles  of  a  strong  current.  The  electrical  sterilization  of  milk 
has  been  patented.  Wines  are  improved  by  electricity.  The  steriliza- 
tion of  drinking  water  by  ozone  is  also  an  application  of  electricity, 
though  of  course  the  ozone  once  formed  by  the  current  acts  as  a  chem- 
ical compound  independently  of  its  source,  and  the  same  effect  would 
be  produced  if  the  ozone  were  manufactured  chemically. 

*  Prepared  by  Otto  Rahn. 


282 


CHAPTER  V 
INFLUENCE'OF  MECHANICAL  AGENCIES* 

PRESSURE. — The  resistance  of  microorganisms  to  mechanical  pres- 
sures is  very  great.  Pressures  of  3,000  atmospheres!  will  not  kill  the 
majority  of  bacteria  in  four  hours.  They  are,  however,  weakened  and 
some  species  will  die.  A  specific  difference  between  the  molds,  yeasts, 
and  bacteria  in  this  particular  does  not  seem  to  exist.  Of  the  organisms 
exposed  to  2,000  atmospheres  for  ninety-six  hours,  Bad.  anthracis,  Bact. 
pseudodiphtheria,  M.  pyogenes  var.  aureus,  Oidium  lactis  and  Saccharo- 
myces  ceremsia  survived,  while  seven  other  organisms  lost  the  power  of 
multiplication.  Some  of  these  were  not  dead,  however,  since  they 
retained  their  motility  for  several  days.  It  is  noteworthy  that  high 
pressure  will  destroy  one  quality  (multiplication)  and  not  affect  another 
(motility).  Pigment-production  and  virulence  of  pathogenic  bacteria 
were  either  diminished  or  lost  completely.  The  resistance  against 
high  pressure  is  necessary  for  the  organisms  which  cause  the  decay 
of  organic  matter  at  the  bottom  of  the  oceans.  Vertebrates  breathe 
oxygen  in  the  form  of  gas  or  have  at  least  an  organ  filled  with  gas  (fish 
bladder) ;  the  volume  of  gas  is  changed  considerably  by  slight  changes 
of  pressure;  this  will  affect  organisms  depending  on  gas.  Microorgan- 
isms do  not  require  gas  as  such.  They  can  absorb  gases  only  in 
solution.  A  change  of  pressure  therefore  will  not  cause  a  change  of 
volume,  since  liquids  have  a  very  small  coefficient  of  compression. 

The  situation  is  entirely  different  if  the  liquid  is  not  exposed  to  the 
pressure  directly,  but  to  compressed  air.  In  this  case,  the  chemical 
effect  of  the  gas  is  the  deciding  agent.  The  higher  the  pressure,  the 
more  gas  will  be  dissolved  in  the  culture  medium.  The  fatal  pressure 
under  these  conditions  will  vary  as  much  as  the  fatal  dose  of  an  antisep- 
tic; it  depends  upon  the  chemical  qualities  of  the  gas,  upon  the  pressure 
(concentration),  upon  the  temperature,  and  upon  the  organism. 

*  Prepared  by  Otto  Rahn. 

fOne  atmosphere  is  i  kg.  pressure  per  square  centimeter  (or  about  15  pounds  per  square 
inch). 

283 


284  PHYSICAL  INFLUENCES 

Some  data  have  been  given  already  in  the  chapter  on  oxygen  require- 
ments. It  was  mentioned  in  that  connection  that  Bad.  butyricum  can- 
not tolerate  more  than  0.65  per  cent  of  the  total  oxygen  content  in  air 
(o. 2  atmosphere) ;  mother  words,  an  oxygen  pressure  higher  than 0.0013 
atmosphere  will  kill  the  organism.  The  maximum  pressure  for  B. 
prodigiosus  was  found  to  be  about  5.4  to  6.3  atmospheres.  Very  few 
experiments  have  been  made  with  other  gases.  Carbon  dioxide  at  a 
pressure  of  50  atmospheres  retards  the  growth  of  bacteria  in  water  and 
will  sterilize  it  in  twenty-four  hours.  Suspensions  of  pure  cultures  of 
B.  typhosus  and  M sp.  comma  are  killed  by  50  atmospheres  carbon  dioxide 
pressure  in  three  hours.  Milk  cannot  be  sterilized  by  this  pressure  but 
bacteria  do  not  multiply.  Carbonated  milk  has  been  recommended  as 
a  refreshing  drink  by  several  investigators.  The  ordinary  market  milk 
will  keep  about  two  days  longer  under  the  pressure  of  10  atmospheres 
(150  pounds)  than  without  pressure.  If  pasteurized  it  is  said  to  keep 
for  a  week. 

GRAVITY. — Gravity  would  have  a  great  influence  upon  the  growth  of 
microorganisms  in  liquids  if  their  specific  gravity  were  much  greater 
than  that  of  water.  This  does  not  seem  to  be  the  case  however.  It  has 
been  estimated  by  accurate  weighing  to  vary  between  1.038  and  1.065. 
Very  much  higher  results  (1.3  to  1.5)  have  been  obtained  by  centrifuging 
bacteria  in  salt  solutions  of  varying  specific  gravity,  but  these  data  are 
not  exact  since  the  salt  solution  will  diffuse  into  the  cells  and  thus  in- 
crease their  weight.  The  specific  gravity  being  very  nearly  that  of  the 
culture  medium,  it  is  plainly  seen  that  gravity  has  but  little  influence. 
The  microorganisms  will  live  suspended  in  the  liquid  and  sediment  out 
very  slowly.  The  slightest  current  in  the  liquid  will  carry  them 
around  and  distribute  them  through  the  medium.  The  motility  is  of 
minor  importance;  the  actual  distance  covered  by  motile  bacteria  has 
been  measured,  and  under  the  most  careful  exclusion  of  currents  in  the 
liquid  has  been  found  to  be  about  a  millimeter  in  a  minute  for  B.  subtilis. 
This  is  very  slow  compared  with  the  speed  of  the  circulating  water 
moved  by  changes  of  temperature  or  other  incidental  agents. 

Yeast  cells  and  other  gas  producers  use  the  carbon  dioxide  as  a  ve- 
hicle. The  gas  bubbling  up  in  the  fermenting  liquid  keeps  it  constantly 
in  motion  and  moves  the  yeast  cells  against  gravity  toward  the  surface 
where  the  gas  escapes  and  lets  the  cells  fall  back  to  the  bottom. 

The  production  of  scums  and  pellicles  on  the  surface  by  organisms 


INFLUENCE   OF   MECHANICAL  AGENCIES  285 


which  are  heavier  than  the  liquid  they  float  on,  is  often  accomplished  by 
small  gas  bubbles  between  the  cells  (Mycoderma).  In  other  instances, 
it  may  be  just  the  floating  of  cells  having  oily  surfaces. 

The  growth  is  influenced  by  gravity  very  little.  The  sporangia  of 
molds  are  the  only  exceptions,  growing  decidedly  away  from  the  center 
of  gravity  (negative  geotropism). 

AGITATION. — For  the  majority  of  microorganisms,  the  quiet,  undis- 
turbed growth  of  the  laboratory  culture  is  the  normal  or  the  ideal  one. 
Such  cultures,  if  shaken  for  a  considerable  time,  show  a  decrease  of  liv- 
ing organisms,  and  it  is  possible  to  sterilize  cultures  by  continued  shak- 
ing. The  effect  is  not  a  simple  mechanical  breaking  or  tearing  of  the 
cells-  The  bacteria  break  up  into  the  finest  particles.  This  is  also  the 
case  if  cultures  are  exposed  for  several  days  to  the  trembling  motion 
caused  by  the  working  of  very  heavy  machines.  There  is  no  grinding  or 
tearing  effect  but  the  cells  break  to  pieces  just  the  same. 

A  slight  and  slow  agitation  seems  to  be  advantageous  for  many  cul- 
tures, only  continuous  heavy  motion  proves  harmful.  Different  organ- 
isms show  wide  variations  in  their  resistance  to  agitation. 


DIVISION  IV 
CHEMICAL  INFLUENCES 


CHAPTER  I 
STIMULATION  OF  GROWTH* 

The  influence  of  chemical  substances  upon  microorganisms  may 
be  helpful  or  harmful,  or  not  noticeable.     As  helpful  must  be  con- 
sidered above  all  the  food  compounds.     Unless  given  in  such  large 
doses  as  to  cause  a  physical  or  osmotic  effect  they  will  stimulate 
the    development.      Other    substances,    not  food,    can   also   act    as 
stimulants.     It  is  a  recognized  fact  of  long  stand- 
ing that  many  poisons  in  very  small  doses  will 
stimulate.      This    applies    to    the    most    highly 
developed  animals  and  plants  as  well  as  to  micro- 
organisms.    Raulin  noticed  in   1869    that   Asper- 
gillus  niger  grew  very  much  better  in  a  nutrient 
solution  if  a  small  amount  of  zinc  salt  was  added. 
He  considered  the  zinc,  therefore,  as  a  necessary 
constituent  of  the  mold  cells.     Alcoholic  fermenta- 
tion can  be  stimulated  by  metallic  salts.     It  is  be- 
' /:  •'  •'  - '  lieved  by  some  physiologists  that,  as  a  law  of  nature, 

FIG.  120.— Chem-     every  substance  that  is  injurious  in  a  certain  con- 

o  taxis.      (After     centra tion  is  a  stimulant  in  a  lower  concentration. 
Fischer.)  ...  .  ..  .        ,        .     , 

A  similar  action  of  certain  chemical  compounds 

upon  enzymes  has  been  noticed,  retarding  in  high  concentrations, 
stimulating  in  weaker  solution. 

CHEMOTROPISM  AND  CHEMOTAXIS. — Microorganisms  manifest  their 
preference  for  certain  foods  not  by  a  stimulated  growth  alone.  They 
also  make  efforts  to  obtain  better  food  by  growing  or  moving  toward  it, 
which  isjiot  a  manifestation  of  a  rudimentary  intellect.  Such  reactions 
of  microorganisms  may  be  accounted  for  largely  by  chemical  or  osmotic 

*•  Prepared  by  Otto  Rahn. 

286 


STIMULATION    OF    GROWTH  287 

forces.  In  a  solid  medium  the  hyphae  of  molds  will  grow  toward  the 
best  source  of  food  supply.  This  growth  on  account  of  chemical 
stimulation  is  called  chemotropism,  analogous  to  the  phototropism 
or  growth  toward  light.  If  some  injurious  compound  is  offered, 
the  hyphae  will  grow  away  from  it.  Thus  we  have  to  distinguish 
between  positive  arid  negative  chemotropism.  The  motile  organisms, 
bacteria  as  well  as  protozoa,  demonstrate  their  preference  for  certain 
food  compounds  by  swimming  toward  them.  This  is  called  chemotaxis 
(Fig.  120).  Here  also  a  positive  and  negative  chemotaxis  must  be 
distinguished,  the  latter  taking  place  if  injurious  substances  are  present. 


CHAPTER  II 

INHIBITION  OF  GROWTH* 
POISONS,  GERMICIDES,  DISINFECTANTS,  ANTISEPTICS,  PRESERVATIVES 

A  great  number  of  inorganic  and  organic  bodies  will  destroy 
life  in  comparatively  weak  solutions.  These  substances  are  called 
poisons  if  they  are  considered  in  their  effect  upon  man  and  animals.  In 
their  application  to  microorganisms  they  are  generally  called  germicides 
(germ-killers),  or  disinfectants  if  the  emphasis  is  laid  upon  the  prevention 
of  infection  rather  than  upon  the  actual  killing  of  the  microorganisms. 
Analogous  to  the  general  term  germicides,  the  terms  bactericide  and 
fungicide  are  used  occasionally.  The  term  antiseptic  means  a  prevention 
of  sepsis  which  may  be  accomplished  by  checking  the  growth  without 
necessarily  killing  all  microorganisms.  The  meaning  of  the  word  pre- 
servative is  practically  the  same,  only  the  latter  is  used  more  commonly 
in  relation  to  foods,  feeding  stuffs  and  preparations  of  similar  origin 
while  the  word  antiseptic  is  largely  used  in  relation  to  microbial  diseases. 
A  strict  line  cannot  be  drawn  between  any  of  these  definitions.  A  dis- 
infectant, if  diluted,  becomes  an  antiseptic.  A  strong  salt  solution  is  an 
antiseptic  for  some  organisms  and  a  disinfectant  for  others.  Of  the 
above  expressions,  germicide  is  the  most  definite,  but  is  not  so  commonly 
used  as  the  others. 

MODE  OF  ACTION. — The  action  of  a  poison  upon  the  cell  is  generally 
considered  an  action  upon  the  protoplasm.  The  poison  is  supposed  to 
combine  chemically  with  the  cell  plasma  producing  compounds  which 
interfere  with  the  continuation  of  the  life  processes  and  thus  cause 
death.  If  the  cell  has  been  subjected  to  the  action  of  the  poison  only  a 
short  time,  it  can  be  saved  by  removing  the  poison.  Bacteria  can  be 
treated  with  mercuric  chloride  (HgCl2)  so  that  they  will  no  longer  de- 
velop if  transferred  to  a  fresh  medium.  If  the  mercuric  chloride  is  re- 
moved from  the  cell  by  means  of  hydrogen  sulphide,  some  of  the  organ- 
isms may  be  revived. 

The  mode  of  death  through  poison  is  the  same  as  that  through 

*  Prepared  by  Otto.Rahn. 

288 


INHIBITION    OF    GROWTH 


289 


heat  or  drying.  The  number  of  cells  dying  in  a  given  time  interval  is 
proportional  to  the  number  of  cells  surviving.  In  the  last  five  years, 
this  has  been  tested  and  found  true  with  practically  all  disinfectants. 
Fig.  1 2 1  shows  the  curves  plotted  from  data  obtained  with  BacL  anthracis, 
the  full-drawn  line  representing  the  number  of  live  spores  in  .21  per 


3500 


3000 
I 


2000 


1500 


IOOO 


500 


o    .» 


10 


FIG.  i2i— Curve  of  disinfection.     Spores  of  Baa.  anthracis  in  mercuric  chloride 
solution.     (After  Chick.) 

cent  of  mercuric  bichloride,  the  dotted  line  the  same  in  .11  per  cent 
solution. 

The  (apparent)  resistance  of  the  few  remaining  cells  is  of  great  im- 
portance in  those  applications  of  disinfection  where  a  thorough  kill- 
ing of  all  bacteria  is  intended,  e.g.,  in  the  treatment  of  drinking  water. 
Our  ideas  of  the  efficiency  of  a  disinfectant  would  depend,  therefore, 

19 


2QO  CHEMICAL  INFLUENCES 

upon  the  accuracy  with  which  we  can  prove  the  presence  of  a  certain 
bacterium. 

FACTORS  INFLUENCING  DISINFECTION. — The  efficiency  of  a  dis- 
infectant depends  upon  several  factors.  Moisture  is  necessary — 
a  dry  poison  has  only  a  very  slow  action  upon  microorganisms.  For 
this  reason,  absolute  alcohol  has  not  nearly  the  same  germicidal  power 
upon  dry  bacteria  as  diluted  alcohol;  the  strongest  poisonous  effect 
is  obtained  by  a  50  to  70  per  cent  solution.  The  necessity  of  moisture 
is  further  demonstrated  in  the  sterilization  with  gases,  as  with  formal- 
dehyde. The  effect  of  formaldehyde  gas  without  the  provision  of  a 
very  moist  atmosphere  is  surprisingly  weak. 

The  temperature  is  also  quite  an  important  factor  in  the  study 
of  disinfectants.  Since  poisoning  is  supposed  to  be  a  chemical  effect, 
it  must  be  expected  that  the  poisoning  process  like  other  chemical 
processes  will  take  place  faster  at  a  higher  temperature.  As  a  matter 
of  fact,  the  death  rate  through  poisoning  is  usually  doubled  or  trebled 
by  a  temperature  increase  of  10°.  Above  the  optimum  temperature, 
where  the  growth  is  not  very  vigorous,  and  when  the  disinfecting 
power  of  the  poison  is  increased  considerably  by  the  higher  temperature, 
a  very  small  amount  of  poison  will  have  a  very  strong  germicidal  effect. 
The  combination  of  high  temperatures  with  a  disinfectant  has  been 
suggested  as  a  means  of  sterilizing  foods.  This  has  been  tried  in 
the  case  of  milk  with  hydrogen  peroxide  at  50°  to  60°. 

It  makes  a  considerable  difference  whether  the  organisms  which 
are  tested  with  a  certain  disinfectant  are  in  a  culture  with  their  food 
material,  or  suspended  in  water  or  salt  solution  without  any  food.  It 
is  very  probable  that  part  of  the  disinfectant  is  acted  upon  by  the  food 
products  which  are  partly  protein  substances  and  are  in  many  ways 
similar  to  the  protoplasm  of  the  bacterial  cells.  It  is  especially  diffi- 
cult to  poison  bacteria  in  blood,  pus,  or  similar  material.  The  sensi- 
bility of  the  microorganisms  in  pure  water  is  remarkable.  Very  small 
doses  which  would  not  be  considered  efficient  under  any  other  condition, 
will  destroy  microorganisms  in  pure  water.  The  concentration  of 
chloride  of  lime  which  is  sufficient  to  sterilize  drinking  water,  does 
not  at  all  suppress  the  development  of  bacteria  in  sewage. 

The  influence  of  the  number  of  cells  is  evident  from  the  above  ex- 
planations of  the  mode  of  action,  and  from  the  curves  of  disinfection. 
The  concentration  of  the  poison  is  of  course  of  greatest  importance. 


INHIBITION   OF   GROWTH  2QI 

Recent  investigations  have  shown  the  rather  unexpected  fact  that  the 
efficiency  of  a  poison  is  not  proportional  to  its  concentration.  If 
a  certain  poisonous  solution  is  diluted  with  an  equal  volume  of  water, 
we  might  expect  it  to  be  half  as  poisonous  as  before,  but  depending 
upon  the  chemical  nature  of  the  poison,  it  may  be  more  poisonous 
than  expected,  or  considerably  less.  It  follows  from  this  that  two  dif- 
ferent poisons  of  the  same  intensity,  if  diluted  in  the  same  proportion, 
may  not  have  the  same  intensity  any  more. 

Microorganisms  will  gradually  become  accustomed  to  certain  poi- 
sons, and  become  more  resistant.  This  principle  has  been  utilized 
in  the  manufacture  of  distilled  alcohol;  yeasts  have  been  cultivated 
which  can  tolerate  a  high  concentration  of  acid;  the  acid  serves 
to  suppress  bacteria  producing  undesirable  fermentations. 

The  age  of  the  culture  and  the  stage  of  development  will  naturally 
change  the  resistance  of  a  species  materially.  The  old  cultures 
which  are  past  the  culmination  of  growth  will  be  much  more  sensitive 
to  any  poison  unless  a  spore-producing  organism  is  under  test.  In 
this  case,  we  find  a  greatly  increased  resistance,  similar  to  the 
increased  resistance  of  spores  against  drying  and  heat. 

THE  CLASSIFICATION  OF  DISINFECTANTS  is  very  difficult  as  long 
as  we  cannot  explain  completely  the  process  of  poisoning.  It  is  im- 
possible to  arrange  them  according  to  the  intensity  of  action,  because 
the  intensity  of  influence  depends  not  only  upon  the  disinfectant, 
but  also  upon  the  species  of  organisms.  Some  yeasts  can  resist  ten 
times  as  much  alcohol  as  certain  bacteria.  Formaldehyde  is  not 
nearly  as  strong  an  agent  with  molds  as  it  is  with  bacteria.  The  dis- 
infectant concentration  of  a  poisonous  substance  is  not  absolute.  The 
simplest  method  of  grouping  is  by  chemical  structure  and  qualities. 
Of  the  following  natural  groups  can  be  distinguished  acids  (inorganic 
and  organic),  metallic  salts,  hydrocarbons  (aliphatic  and  cyclic), 
alcohols  (aliphatic ' and  cyclic),  aldehydes,  anaesthetics,  essential  oils, 
oxidizing  agents  and  reducing  agents. 

The  first  three  groups,  acids,  alkalies  and  salts,  are  distinguished 
from  the  rest  as  electrolytes;  the  strength  of  acids  and  alkalies  (chemic- 
ally speaking)  is  measured  by  the  degree  of  electrolytic  dissociation. 
The  disinfectant  value  follows  largely  the  same  law.  The  strongest 
acids  in  the  chemical  sense  are  also  the  strongest  disinfectants.  There 
are  exceptions,  however,  where,  besides  the  poisonous  effect  due  to 


2Q2  CHEMICAL   INFLUENCES 

the  degree  of  dissociation,  there  is  a  specific  effect  due  to  the  chemical 
structure,  as  is  the  case  of  nitrous,  salicylic  and  hydrocyanic  acids. 
The  same  is  true  of  alkalies.  With  metallic  salts,  the  action  will  depend 
mainly  upon  the  metal  in  solution,  but  the  electrolytic  dissociation 
is  also  of  importance.  NaCl  will  decrease  the  dissociation  of  mer- 
curic chloride  (HgC^)  and  decrease  also  its  disinfectant  power.  Mer- 
curic chloride  dissolved  in  absolute  alcohol  is  not  dissociated.  In 
this  case,  it  has  almost  no  action  upon  bacteria. 

Acids  are  not  commonly  used  as  disinfectants,  except  in  the  house- 
hold, but  they  play  a  certain  role  in  nature.  The  common  fruits  con- 
tain so  much  acid  that  bacteria  cannot  easily  attack  them;  the  decay- 
ing of  fruit  is  almost  exclusively  due  to  molds  which  have  a  preference 
for  acid  media.  The  acid  in  the  stomach  of  man  and  animals  plays  an 
important  role  as  a  sterilizing  agent  for  the  food.  Many  microorgan- 
isms are  killed  in  the  stomach.  In  the  household,  the  natural 
acidity  of  fruit  helps  in  keeping  canned  fruit,  preserves  and  jellies. 
Especially  in  heating,  the  acid  together  with  the  high  temperature 
has  a  very  strong  germicidal  effect.  Vinegar  is  often  used  to  pre- 
serve fruit  and  vegetables;  in  some  parts  of  the  country,  meat  is  kept 
in  buttermilk.  Benzoic  and  salicylic  acids  are  often  used  in  the  pres- 
ervation of  fruit  and  vegetables.  Their  poisonous  influence  is  not 
so  much  due  to  the  acid  reaction  but  to  the  specific  chemical  character 
of  these  compounds. 

Of  the  alkalies,  only  one  is  used  extensively,  namely,  lime;  quick- 
lime (CaO)  is  considered  a  valuable  disinfectant  for  excreta  in  privy 
vaults;  it  is  universally  applied  as  a  whitewash  in  stables,  barns, 
poultry  houses  and  similar  buildings.  Quite  commonly,  it  is  used  as 
"milk  of  lime"  (one  part  of  slaked  lime  with  four  parts  of  water). 
It  should  be  kept  in  mind  that  the  calcium  oxide  unites  with  the  carbon 
dioxide  of  the  air  and  thus  gradually  loses  its  disinfecting  power. 

Of  the  metallic  salts,  many  are  well-known  germicides.  The  most 
powerful  disinfectant  is  mercuric  chloride  (HgCU)  which  is  one  of  the 
standard  disinfectants.  It  is  generally  used  in  a  dilution  i :  1000 
which  is  sufficient  to  kill  all  vegetative  cells  as  well  as  spores  in  a  few 
minutes.  Quite  commonly,  hydrochloric  acid  or  salt  is  added,  to 
prevent  coagulation  or  precipitation  of  slimy  or  albuminous  matter 
which  would  protect  the  enclosed  bacteria  from  immediate  contact 
with  the  poison.  The  addition  of  hydrochloric  acid  or  any  chloride 


INHIBITION   OF   GROWTH  293 

decreases  somewhat  the  disinfectant  value  for  bacteria  suspended  in 
distilled  water  because  it  decreases  the  electrolytic  dissociation. 

Another  disinfectant  of  remarkable  strength  is  silver  nitrate;  it 
is  not  used  commonly  because  of  its  high  price.  It  also  decomposes 
easily  and  leaves  dark  spots  on  the  skin  and  clothes.  Of  the  other 
metallic  salts,  copper  and  iron  sulphate  are  not  used  extensively, 
though  recommended  for  the  disinfection  of  feces.  Zinc  sulphate  may 
be  applied  to  mucous  membrane  the  same  as  silver  nitrate.  Many 
other  salts  may  be  used  occasionally  for  disinfecting  purposes,  though 
the  expense  or  undesirable  qualities  prevent  their  common  application. 

The  alcohols  are  well  known  for  their  poisonous  effects,  but  the 
value  of  ethyl  alcohol  as  a  disinfectant  is  usually  overestimated.  It 
takes  quite  strong  alcoholic  solutions,  more  than  20  per  cent,  to  kill 
certain  yeasts  and  the  spores  of  some  bacteria  in  less  than  a  day, 
and  a  complete  sterilization  by  alcohol  in  a  few  minutes  cannot  al- 
ways be  guaranteed  even  with  50  to  60  per  cent  solution.  It  has 
already  been  mentioned  that  desiccated  organisms  are  very  resistant 
to  concentrated  alcohol,  more  so  than  to  a  50  per  cent  mixture. 
Methyl  alcohol  is  weaker,  the  higher  alcohols,  especially  amyl  alcohol, 
are  stronger  disinfectants  than  ethyl  alcohol.  They  all  give  good 
results  in  the  presence  of  water  while  the  absolute  alcohols  have 
scarcely  any  effect  upon  desiccated  bacteria.  None  of  these  alcohols 
in  whatever  concentration  they  may  be  used,  can  be  relied  upon  to 
kill  bacterial  spores. 

Stronger  germicidal  effects  can  be  obtained  by  the  alcohols  of  the 
benzol  group,  of  which  phenol  or  so-called  carbolic  acid  (CeH6OH) 
is  the  simplest  representative.  Phenol,  like  ethyl  alcohol,  is  not  as 
effective  as  is  commonly  believed.  It  is  applied  in  solutions  from  .5  per 
cent  to  5  per  cent  ordinarily,  but  it  usually  takes  a  long  time  even  for 
the  5  per  cent  solution  to  kill  vegetative  cells  as  Bact.  tuberculosis  or 
B.  coli;  it  is  inefficient  against  anthrax  spores.  More  powerful  are 
the  higher  cyclic  alcohols,  of  which  the  cresols  are  examples.  They  are 
used  extensively  as  disinfectants  and  antiseptics.  They  are,  together 
with  phenol,  coal-tar  constituents  and  are  sold  commercially  under  many 
different  names,  either  pure  or  mixed  with  soap  or  other  disinfectants 
which  make  them  emulsify  readily  in  water.  The  cresols  are  almost 
insoluble  in  water,  and  not  as  effective  in  solutions  as  they  are  in 


294  CHEMICAL  INFLUENCES 

emulsions.  The  disinfecting  properties  of  tar  come  from  the  cresol 
contained  in  it, 

Hydrocarbons  are  used  only  for  laboratory  experiments  as  very 
weak  antiseptics.  The  aliphatic  bodies,  as  methane,  etc.,  which  con- 
stitute a  large  part  of  coal  gas,  have  very  little  if  any  effect  upon  bac- 
teria; gas  is  used  occasionally  in  place  of  hydrogen  for  growing  anae- 
robic bacteria.  Benzol,  xylol,  and  toluol  are  antiseptics,  if  shaken 
frequently  with  the  liquid  to  be  protected,  but  they  are  not  reliable 
as  disinfectants.  The  same  is  true  with  the  common  anaesthetics,  ether 
and  chloroform.  The  high  prices  of  these  agents  forbid  their  general 
use,  but  they  are  sometimes  used  for  laboratory  work. 

The  essential  oils  have  a  little  more  practical  importance.  Some  of 
these  are  the  main  constituents  of  mouth  washes,  especially  the  oil  of 
peppermint  (menthol),  of  thyme  (thymol),  and  of  eucalyptus  (eucalyp- 
tol).  Their  action  is  very  weak,  however.  The  volatile  oils  of  spices 
have  to  be  considered  in  the  preserving  of  fruit,  pickles,  catsups,  and 
other  food  products.  Though  the  antiseptic  value  in  general  is  insigni- 
ficant, certain  microorganisms  are  sensitive  to  certain  spices.  The 
bacteria  of  the  mesentericus  group  are  said  to  be  suppressed  entirely 
by  quite  small  quantities  of  garlic,  while  others,  like  the  lactic  bacteria, 
are  not  affected  at  all.  Cloves,  cinnamon  and  allspice  are  the  most 
efficient  spices,  while  the  disinfectant  power  of  black  and  white  pepper 
and  mustard  is  very  small. 

The  most  important  disinfectant  has  not  been  mentioned,  because 
it  does  not  belong  to  any  of  the  above  groups.  This  is  formaldehyde. 
Formaldehyde  (HCOH)  is  a  gas,  soluble  in  water  to  the  amount  of  40 
per  cent  at  room  temperature;  it  does  not  attack  metal,  clothing,  wood- 
work, and  is,  therefore,  preferable  to  many  other  disinfectants  for  steril- 
izing rooms.  It  kills  spores  of  bacteria  in  a  short  time  in  a  i :  1000  di- 
lution. Its  greatest  importance  lies,  however,  in  its  gaseous  nature, 
because  it  can  be  applied  to  rooms  and  buildings  by  simply  evaporating 
it.  The  saturated  40  per  cent  solution  can  be  evaporated  directly  or  by 
generating  steam  which  passes  through  the  formaldehyde  solution;  this 
latter  method  has  the  advantage  of  saturating  the  air  with  moisture, 
which  increases  the  power  of  the  formaldehyde  gas.  Formaldehyde 
can  also  be  obtained  in  a  dry  form;  it  polymerizes  to  a  white  crystalline 
substance,  paraformaldehyde  ((HCOH)3)  which  can  be  changed  back  to 
formaldehyde  gas  by  gentle  heating.  This  paraformaldehyde  is  com- 


INHIBITION   OF   GROWTH  2  95 

monly  used  instead  of  the  liquid,  because  it  is  more  easily  handled  and  is 
quite  inoffensive  in  its  solid  form,  while  the  formaldehyde  solution  has  a 
very  penetrating  odor  and  is  exceedingly  harmful  to  the  mucous  mem- 
brane of  the  respiratory  organs. 

Of  the  oxidizing  agents,  oxygen  itself  has  already  been  mentioned. 
Though  it  is  able  to  destroy  certain  anaerobic  bacteria,  it  cannot  be 
called  a  disinfectant.  For  this  purpose,  oxygen  must  be  activated;  such 
oxygen  can  be  obtained  in  the  form  of  ozone  (Os).  It  is  formed  in  air 
under  the  influence  of  electric  discharges  and  can  be  produced  at  a  price 
low  enough  to  allow  its  application  for  use  in  the  sterilization  of  water. 
It  has  also  been  recommended  for  preservation  of  milk. 

Hydrogen  peroxide  (H2O2)  resembles  ozone  in  its  chemical  reactions; 
it  changes  readily  to  H2O  -f-  O,  and  this  oxygen  atom  in  the  nascent 
state  is  quite  effective  as  an  oxidizing  agent.  For  an  antiseptic,  it  must 
be  used  in  at  least  a  i  per  cent  solution,  and  for  an  absolutely  reliable  dis- 
infectant a  still  higher  concentration  is  required.  It  loses  its  disinfect- 
ing property  easily  because  it  is  decomposed  readily  by  the  peroxidases 
of  tissues  and  organic  liquids  as  blood,  milk,  and  pus.  It  is  used  in  the 
preservation  of  milk.  Hydrogen  peroxide  is  slowly  decomposed  by  the 
katalase  of  milk  thus  disappearing  completely. 

Chlorine  in  its  gaseous  form  is  not  used  as  a  disinfectant,  though  its 
germicidal  power  is  quite  strong.  The  so-called  "chloride  of  lime," 
manufactured  by  absorbing  chlorine  in  slaked  lime,  gives  in  water 
hypochlorite  and  free  chlorine;  these  substances  are  good  germicides 
and  chloride  of  lime  is  used  in  the  disinfection  of  privy  vaults,  and  other 
places  in  which  it  may  be  employed  without  injury.  Hypochlorite  is 
now  used  with  great  success  for  rendering  safe  drinking  water  and 
sewage;  it  has  also  become  the  basis  of  some  commercial  dis- 
infectants. 

Potassium  permanganate  is  only  incidentally  used  as  a  disinfectant. 
Its  chemical  qualities  prevent  an  ordinary  use. 

Sulphurous  acid,  or  sulphur  dioxide  (802)  was  for  a  long  time  a 
standard  disinfectant  and  is  still  used  occasionally  for  fumigating  rooms, 
stables,  barns  and  out-buildings  though  it  is  substituted  more  and  more 
by  formaldehyde  which  can  be  applied  almost  as  easily.  The  burning 
of  sulphur  is  an  extremely  simple  process,  but  it  requires  a  moist  air  to 
disinfect  properly,  and  under  these  circumstances  it  will  attack  metal, 
dyes  of  clothing  and  even  the  fiber  itself. 


2p6  CHEMICAL   INFLUENCES 

In  addition  to  these  disinfectants  which  are  used  outside  of  the 
human  body,  or  applied  to  its  surface  only,  there  have  come  into  use 
during  recent  years,  several  disinfectants  which  are  injected  into  the 
body  to  kill  the  microorganisms  in  the  blood.  Among  these  might 
be  mentioned  the  colloidal  metals,  mainly  colloidal  silver  which  is  sold 
under  various  trade  names,  e.g.,  collargol.  It  is  given  especially  in 
pneumonia,  but  its  action  upon  the  bacteria  directly  is  very  insignificant, 
though  it  greatly  stimulates  phagocytosis.  Further,  there  is  to  be 
mentioned  ethoxyl,  given  against  the  protozoon  of  sleeping  sickness, 
and  the  latest  and  most  discussed  of  all,  salvarsan,  an  organic  arsene 
compound,  against  syphilis. 


DIVISION  V 
MUTUAL  INFLUENCES* 


INTRODUCTION 

The  biological  relations  of  microorganisms  are  of  the  greatest  im- 
portance in  nature.  Pure  cultures  in  nature  are  very  rare  and  of  excep- 
tional occurrence;  they  are  hardly  ever  found  except  in  certain  diseases 
of  man,  animals  and  plants.  Generally,  nature  works  with  mixed  cul- 
tures. All  natural  fermentations,  decompositions  and  putrefractions 
are  accomplished  by  a  number  of  different  species  among  which  perhaps 
one  dominates,  but  is  influenced  by  the  rest.  The  study  of  the  mutual 
relations  of  microorganisms  is  in  the  very  first  stage  as  yet;  practically 
all  laboratory  work  is  done  with  pure  cultures.  The  experiences  obtained 
with  pure  cultures  are  not  sufficient  to  explain  all  microbial  activity  in 
nature. 

There  are  many  possibilities  of  mutual  influence  between  different 
organisms.  Generally  three  main  cases  are  distinguished:  symbiosis, 
where  two  organisms  profit  by  the  combination;  melabiosis,  where  one 
profits  by  the  other's  action  without  benefiting  the  other  in  return,  and 
antibiosis,  where  one  organism  injures  the  other.  These  cases  cannot  be 
separated  strictly.  The  relations  are  not  always  constant  through  the 
entire  development  of  the  cultures;  an  originally  beneficial  influence 
may  change  to  an  injurious  one  in  a  few  days.  Many  terms  have  been 
coined  to  designate  all  these  various  possibilities,  but  in  order  to  avoid 
this  multiplicity  of  more  or  less  indefinite  names  for  the  various  relations, 
the  general  term  "association"  has  come  into  use,  especially  when  the 
relationship  is  not  well  understood. 

SYMBIOSIS 

Symbiosis  is  not  very  common  among  microorganisms,  and  it  is 
difficult  to  find  examples  where  true  symbiosis  exists  through  the  entire 

*  Prepared  by  Otto  Rahn. 

2Q7 


298  MUTUAL  INFLUENCES 

development  of  both  organisms.  The  association  of  lactic  bacteria  and 
Oidium  lactis  in  milk  is,  for  a  certain  period  at  least,  a  symbiosis.  The 
bacterium  will  produce  only  a  certain  amount  of  acid,  and  then  it  can 
grow  no  more  because  the  acid  is  too  strong;  the  mold  will  destroy  the 
acid  and  thus  gives  the  bacterium  a  chance  for  continued  activity.  The 
bacterium  produces  the  acid  which  the  mold  likes;  the  mold  in  turn 
removes  the  excess  acid  which  otherwise  would  check  the  bacterial 
activity. 

True  symbiosis  is  more  common  in  the  relation  of  microorganisms 
with  higher  plants  and  animals.  The  standard  example  in  the  plant 
kingdom  is  Ps.  radicicola  in  the  nodules  of  legumes,  feeding  on  carbo- 
hydrates provided  by  the  plant  and  furnishing  the  plant  nitrogen  from 
the  air  which  the  plant  cannot  assimilate  directly.  The  typical  exam- 
ple in  the  animal  kingdom  is  B.  coli  in  the  intestine  of  animals,  being 
nourished  by  the  food  of  the  animal  and  rendering  the  food  more  easily 
digestible. 

METABIOSIS 

Metabiosis  may  be  considered  a  one-sided  symbiosis;  two  organisms 
live  together,  but  only  one  is  benefited,  the  other  remains  uninfluenced 
or  later  may  be  injured  by  the  association;  the  latter  case  is  the  most 
common.  In  this  relation,  one  usually  prepares  the  food  for  the  other. 
It  has  previously  been  mentioned  that  the  metabolic  products  of  one 
species  serve  as  food  for  another  species,  thus  breaking  up  the  various 
organic  compounds  step  by  step  to  smaller  and  simpler  molecules. 
Quite  commonly,  each  step  is  accomplished  by  a  different  species  of 
microorganism.  Consequently,  metabiosis  is  a  very  common  occurrence 
among  microorganisms. 

The  classical  example  is  the  two  nitrifying  bacteria:  the  nitrate  bac- 
terium is  unable  to  oxidize  ammonia,  and  depends  entirely  upon  the  ni- 
trite bacterium  to  oxidize  the  ammonia  to  nitrite;  then,  and  only  then, 
can  the  nitrite  bacterium  grow. 

The  relation  between  yeasts  and  acetic  bacteria  is  also  very  well 
known.  The  yeast  ferments  the  sugar  to  alcohol,  and  then  the  acetic 
organisms  oxidize  the  alcohol  to  acetic  acid.  The  yeast  is  in  no  way 
helped  by  the  acetic  bacteria,  while  these  could  not  form  acetic  acid 
from  sugar  readily.  These  bacteria  depend  upon  the  action  of  the 
alcohol-forming  yeast.  Other  cases  of  metabiosis  are  found  in  the 


ANTIBIOSIS  299 

association  of  lactic  bacteria  with  certain  protein  destroying  organisms. 
The  lactic  bacteria  often  develop  much  better  if  the  protein  bacteria 
grow  together  with  them  or  have  grown  previously  in  milk.  Meta- 
biosis  does  not  require  the  growth  of  the  two  associated  organisms  at 
the  same  time.  The  effect  will  be  the  same  if  first  the  one  and  later  the 
other  develops,  and  even  after  the  first  organism  is  killed  or  removed, 
its  effect  upon  the  pure  culture  of  the  second  will  still  be  noticed.  This 
does  not  occur  in  the  case  of  symbiosis. 

One  species  can  favor  the  development  of  another  by  other  means 
than  food  provision  or  preparation.  Certain  bacteria  cannot  live  in 
acid  media,  and  molds  or  mycodermas  destroying  the  acid  will  render 
possible  the  growth  of  these  bacteria  though  they  do  not  provide  them 
with  food.  This  is  the  case  in  the  ripening  of  certain  soft  cheeses. 
Another  example  is  the  production  of  heat  by  fermenting  organisms  in 
manure,  hay,  ensilage,  enabling  the  development  of  thermophile  organ- 
isms. A  very  interesting  and  important  problem  is  the  growth  of  strictly 
anaerobic  bacteria  near  the  surface  of  liquids  in  association  with 
some  aerobic  bacteria.  How  this  is  really  possible  cannot  be  satisfac- 
torily explained.  Though  the  aerobic  bacteria  continuously  remove  the 
oxygen  from  the  water  a  certain  amount  will  remain,  sufficient  to  pre- 
vent the  growth  of  the  anaerobic  bacteria  under  ordinary  conditions. 
There  seems  to  be  a  certain  protective  influence  derived  from  the  aerobic 
bacteria,  the  nature  of  which  is  unknown. 

ANTIBIOSIS 

The  standard  examples  of  antibiosis  are  the  alcohol  production  by 
yeast  in  sugar  solutions  and  the  acid  production  by  lactic  bacteria  in 
milk.  Fresh  cider  contains  a  large  number  of  bacteria,  yeasts  and 
molds;  some  of  these  organisms  cannot  develop  in  the  acid  medium, 
but  many  will  begin  to  grow.  Some  of  the  bacteria  will  produce  or 
destroy  acid,  others  may  begin  to  work  on  the  nitrogenous  material  of 
the  cider,  and  the  yeasts  produce  alcohol  and  carbon  dioxide.  The 
carbon  dioxide  will  soon  saturate  the  cider  and  begin  to  bubble  up,  thus 
removing  the  other  gases.  The  molds  will  stop  growing  if  the  oxygen 
is  taken  away,  but  some  of  the  bacteria  may  continue  growing  until 
the  alcohol  concentration  checks  their  further  development.  They 
first  cease  to  grow,  then  cease  to  produce  acid  and  finally  die,  while  the 
yeast  is  still  continuing  in  the  fermentation. 


300  MUTUAL   INFLUENCES 

In  the  lactic  fermentation  of  milk,  Bact.  lactis  acidi  combats  all 
other  organisms  by  a  rapid  production  of  lactic  acid.  Though  it  is  pres- 
ent in  fresh  milk  only  in  very  small  numbers,  its  rapid  growth  and  the 
formation  of  acid  which  will  check  and  even  kill  most  other  bacteria 
soon  makes  it  the  dominant  organism  in  the  flora  of  milk,  and  at  the 
time  of  curdling,  it  is  often  difficult  to  find  any  other  organisms 
besides  the  lactic  bacteria.  In  the  preceding  chapter  was  mentioned 
the  metabiosis  of  certain  protein-digesting  bacteria  with  Bact.  lactis 
acidi.  This  metabiosis  can  be  considered  as  such  only  from  the  stand- 
point of  the  lactic  organism.  The  protein  bacteria  are  killed  by  the 
acid  formed  by  the  rapidly  growing  lactic  bacteria.  From  the  view- 
point of  the  protein  bacteria,  the  relation  is  antibiosis.  Another  illus- 
tration of  antibiosis  is  the  acetic  fermentation.  The  formation  of 
acetic  acid  prevents  the  development  of  all  bacteria  and  of  most  yeasts 
and  molds. 

In  all  these  cases,  the  deciding  agent  is  a  well-known  chemical  com- 
pound. In  other  combinations,  the  principle  is  unknown.  Bact.  lactis 
acidi  will  check  the  growth  of  B.  subtilis  not  only  in  milk  where  it  forms 
acid,  but  also  in  sugar-free  broth  where  acid  production  is  impossible. 
Acetic  bacteria  act  upon  the  yeast  cells  not  only  by  means  of  the  acetic 
acid  produced,  but  also  by  some  other,  unknown  agent,  since  vinegar 
is  more  injurious  than  the  corresponding  amount  of  pure  acetic  acid  in 
water.  A  very  remarkable  organism  is  Ps.  pyocyanea;  it  secretes  a 
substance,  pyocyanase,  which  will  kill  and  dissolve  the  cells  of  other 
bacteria  rapidly. 

Parasitism,  which  would  be  classified  under  antibiosis,  has  not  been 
found  to  exist  among  bacteria  or  yeasts;  but  we  know  of  cases  where  one 
mold  grows  on  the  other;  this  is  especially  true  with  the  largest  represen- 
tatives of  the  mucor  family,  which  are  often  attacked  and  sometimes 
killed  by  smaller  fungi. 

RELATIONS  BETWEEN  CELLS  OF  THE  SAME  SPECIES 

That  cells  of  the  same  species  will  also  influence  each  other,  may  well 
be  assumed.  The  simplest  relation  will  be  the  competition  for  food. 
This  will  be  the  case  in  nature  more  commonly  than  in  laboratory  media 
which  are,  as  a  rule,  so  rich  in  nutrients  that  development  ceases  before 
all  food  is  used  up. 


RELATION  BETWEEN  CELLS  301 

The  cause  for  cessation  of  growth  in  a  culture  is  of  great  theoretical 
and  practical  interest.  Apparently  there  are  various  factors  concerned 
in  this.  Lack  of  food,  or  of  one  single  essential  food  compound,  may  be 
the  cause.  This  is  found  sometimes  in  media  where  it  would  be  least 
expected.  Some  strains  of  Strept.  lacticus  are  supposedly  limited  in 
milk  by  the  lack  of  available  nitrogen;  they  cannot  attack  casein  readily 
and  albumin;  besides  these  proteins,  nitrogen  compounds  are  not  plenti- 
ful. Addition  of  peptone  increases  the  maximum  number  of  cells  from 
0.7  billion  to  2.5  billions  per  c.c.  More  commonly,  however,  growth 
is  checked  by  the  accumulation  of  metabolic  products.  Yeasts  are 
checked  by  the  alcohol,  and  acid-formers  by  the  acid,  urea  bacteria 
by  the  alkali.  In  many  of  these  cases,  the  removal,  or  neutralization, 
of  the  inhibiting  product  will  bring  about  new  development. 

The  harmful  products  accumulating  are  not  always  of  such  simple 
nature.  Some  very  interesting  observations  have  been  made  during  the 
last  ten  years.  Eijkmann,  as  the  first,  found  that  B.  coli  reached  its 
maximum  growth  in  gelatin  at  37°  in  a  few  days,  and  that  this  gelatin, 
after  hardening  at  20°,  would  not  support  growth  after  streaking  with 
a  young  culture  of  the  same  organism;  but  after  this  gelatin  had  been 
heated  at  60°  for  half  an  hour,  B.  coli  grew  on  it  as  well  as  on  fresh 
gelatin.  Broth  in  which  B.  coli  had  grown  became  fit  again  for  growth 
of  the  same  bacillus  after  filtration  through  porcelain.  The  inhibition 
of  growth  is,  in  this  case,  due  to  a  compound  which  resembles  a  toxin 
in  many  respects.  The  importance  of  such  investigations  to  general 
physiology  is  evident. 


PART  III 

APPLIED  MICROBIOLOGY 


DIVISION  I* 
MICROBIOLOGY  OF  AIR 


CHAPTER  I 

THE  MICROORGANISMS  OF  THE  AIR  AND  THEIR  DISTRI- 
BUTION 

The  atmosphere  is  not  the  normal  habitat  of  bacteria,  for  growth  and 
multiplication  cannot  take  place  in  it  under  ordinary  conditions.  The 
phrase  "microorganisms  of  the  air"  is  therefore  somewhat  ambiguous. 
The  small  size  of  microorganisms  enables  them  to  remain  suspended  for 
considerable  periods  when  physical  forces  have  separated  them  from  the 
substrata  on  which  they  have  developed. 

MICROORGANISMS  PRESENT  IN  THE  AIR. — Molds,  bacteria,  and  yeasts 
are  all  found  in  the  air  under  certain  conditions.  The  first  two  are  usu- 
ally relatively  abundant,  the  latter  are  less  common. 

The  common  molds  have  adapted  themselves  for  the  most  part  to 
wind  distribution.  They  bear  spores  that  are  small  in  size  and  possess  a 
surface  that  is  not  readily  moistened.  These  spores  are  resistant  to 
desiccation  and  light  and  remain  viable  for  a  considerable  time  even 
under  unfavorable  conditions.  Furthermore,  the  fruiting  bodies  of 
many,  though  not  all  molds,  show  a  distinct  negative  hydrotropism,i.e., 
the  mycelium  remains  in  contact  with  the  moist  substratum  while  the 
threads  which  bear  the  spores  rise  at  right  angles  to  it.  These  latter  are 
so  sensitive  that  they  can  detect  slight  differences  in  the  moisture  con- 
tent of  the  air  and  grow  in  the  direction  which  will  bring  the  spores  into 

•Prepared  by  R.   E.  Buchanan. 

303 


304  MICROBIOLOGY   OF   AIR 

the  driest  situations.  A  slight  current  of  air  will  detach  the  spores  from 
these  structures  and  carry  them  long  distances. 

Bacteria  and  yeasts  lack  the  specific  adaptations  for  wind  distribu- 
tion found  in  molds.  The  material  upon  which  they  have  been  growing 
must  be  dried  and  pulverized  before  they  can  be  blown  about.  Many 
species  produce  spores  or  other  resistant  cells,  and  physiologically  are  as 
well  adapted  for  air  distribution  as  are  the  molds. 

OCCURRENCE  IN  THE  AIR. — Microorganisms  are  found  free  in  the 
air,  attached  to  particles  of  dust,  or  enclosed  in  minute  drops  of  water. 
Mold  spores  are  commonly  free  or  in  unattached  clusters.  Bacteria  and 
yeasts  are  usually  associated  with  dust  particles,  frequently  the  pulver- 
ized substratum  on  which  they  have  been  growing.  Not  all  dust  par- 
ticles have  living  organisms  attached.  It  has  been  computed  that  in 
the  air  of  London  during  a  fog  there  is  only  one  living  organism  for  over 
thirty-eight  millions  of  dust  particles.  Microorganisms  are  some- 
times sprayed  into  the  air  with  water.  Droplets  containing  bacteria 
are  thrown  off  in  the  saliva  in  coughing  or  in  speaking,  and  from  the 
surface  of  fermenting  liquids  on  which  bubbles  are  bursting.  When 
the  drop  is  small  enough,  the  air  currents  keep  it  in  suspension  and  the 
water  soon  evaporates  and  frees  the  organism.  This  brings  about  the 
condition  first  discussed,  free  bacteria  in  the  air.  The  decrease  in 
weight  and  size  incident  to  this  loss  of  water  probably  accounts  for  the 
fact  that  the  so-called  "infectious  droplets"  are  sometimes  carried  for 
considerable  distances. 

How  MICROORGANISMS  ENTER  THE  AIR. — In  comparatively  few  in- 
stances do  microorganisms  possess  mechanical  devices  for  projecting 
the  spores  or  other  cells  into  the  air  for  wind  distribution.  Usually  the 
organism  is  passive  and  is  freed  only  by  air  currents  or  by  mechanical 
agitation.  Some  molds,  as  has  been  stated,  release  their  spores  even  in 
the  presence  of  moisture,  so  that  complete  desiccation  is  unnecessary  for 
their  dispersal.  Bacteria  and  yeasts,  on  the  other  hand,  are  not  usually 
given  off  from  moist  surfaces.  Only  when  dry  and  pulverized  can  the 
bacterial  medium  be  readily  blown  about.  Hansen  found  that  in  the 
immediate  vicinity  of  a  heap  of  decaying  malt,  the  air  was  comparatively 
free  from  bacteria.  Winslow  has  shown  that  sewer  air  is  frequently 
practically  free  from  bacteria  although  the  surface  with  which  it  comes 
in  contact  teems  with  bacterial  life.  Mechanical  agitation  often  throws 
large  numbers  of  organisms  into  the  air.  Moving  hay  and  straw, 


THE   MICROORGANISMS    OF   THE   AIR  305 

grooming  animals,  sweeping  a  floor  or  carpet  will  multiply  the  dust  and 
bacterial  content  of  the  air  many  times.  In  a  similar  manner,  tiny, 
germ-holding  droplets  may  be  scattered  by  the  splashing  of  sewage  or  of 
fermenting  or  putrefying  liquids,  and  in  speaking,  sneezing  or  coughing. 

CONDITIONS  FOR  SUBSIDENCE  OF  BACTERIA. — The  length  of  time 
during  which  an  organism  may  remain  suspended  in  the  air  is  dependent 
upon  several  factors.  Small  particles  settle  out  more  slowly  than  large 
for  the  reason  that  as  the  size  of  an  object  is  decreased,  the  surface  area 
decreases  less  rapidly,  proportionately,  than  the  volume.  The  lifting 
effect  of  air  currents  depends  upon  the  ratio  of  surface  area  to  volume 
and  specific  gravity.  The  smaller  the  object,  therefore,  the  greater  is 
the  resistance  to  subsidence  Consequently,  bacteria  usually  settle 
out  of  air  very  slowly  if  free  in  a  quiet  atmosphere.  The  time  of  sus- 
pension is  determined  also  by  the  velocity  of  the  air  currents.  While 
considerable  velocity  may  be  necessary  to  dislodge  microorganisms  and 
bring  them  into  suspension,  a  very  slight  air  current  will  sustain 
them.  Winslow  has  found  that  a  current  of  17  inches  per  minute  is 
sufficient  to  sustain  B.  prodigiosus.  The  relative  humidity  of  the 'air  is 
also  an  important  factor.  In  a  supersaturated  air  solid  particles,  such 
as  bacteria,  become  foci  of  condensation  for  water  and  quickly  settle 
out.  When  dust  is  present  in  considerable  quantities,  and  certain  elec- 
trical or  moisture  conditions  exist,  flocculation  occurs  and  the  larger 
bodies  so  formed  subside  rapidly.  The  character  and  abundance  of 
surfaces  with  which  the  suspended  particles  may  come  in  contact  also 
play  an  important  part.  Moist  surfaces  are  much  more  effective  in 
retaining  particles  than  those  which  are  dry. 

DETERMINATION  OF  THE  NUMBER  OF  BACTERIA  IN  THE  AIR. — The 
number  of  bacteria  in  the  air  is  frequently  determined  by  exposing  open 
petri  dishes  of  gelatin  or  agar  in  different  places  for  definite  periods. 
This  is  a  comparative  quantitative  method  only.  The  number  of  colo- 
nies developing  upon  these  plates  will  give  the  number  of  dust  particles 
having  living  spores  or  cells  upon  them  that  fall  in  the  given  area  under 
the  conditions  of  the  experiment.  Evidently  this  is  of  value  only  for 
rough  comparative  work  as  constantly  shifting  currents  of  air  usually 
introduce  great  errors.  A  somewhat  more  accurate  method  is  to  draw 
measured  volumes  of  air  into  a  flask,  the  bottom  of  which  is  covered 
with  a  layer  of  gelatin  or  agar.  The  colonies  which  develop  represent 
the  number  of  organisms  which  settle  out  from  the  given  volume.  More 

20 


306  MICROBIOLOGY  OF  AIR 

accurate  results  still  may  be  obtained  by  drawing  measured  vol- 
umes of  air  in  small  bubbles  through  liquid  gelatin.  Practically  all  of 
the  particles  will  be  retained  and  the  number  of  colonies  which  develop 
may  be  counted.  This  method  is  sometimes  modified  by  drawing  the 
air  through  a  definite  volume  of  water,  care  being  taken  to  insure  suffi- 
cient contact  of  air  and  water  to  remove  all  dust  particles.  A  propor- 
tionate part  of  the  water  is  then  plated  and  the  number  of  organisms 
estimated.  Air  is  sometimes  drawn  through  a  filter  made  of  sugar, 
sodium  sulphate,  or  sodium  chloride,  and  this  material  then  dissolved 
in  water  and  plated.  Sand,  asbestos,  glass,  etc.,  are  sometimes  used 
as  air  filters,  then  thoroughly  washed,  and  the  wash  water  plated. 

Relative  quantitative  examination  of  the  air  is  of  more  historical 
than  practical  importance.  It  has  been  useful  in  the  development  of 
the  germ  theories  of  fermentation  and  of  disease  and  in  overthrowing 
the  theory  of  spontaneous  generation.  There  is  so  little  ordinarily  to  be 
learned  by  a  study  of  the  air  flora  that  a  comparison  of  plates  exposed 
directly  will  usually  suffice.  Where  more  accurate  results  are  desired, 
one  must  resort  to  one  of  the  filtration  methods  discussed  above. 

Qualitative  determinations  of  the  species  of  air  organisms  are  not 
often  made.  When  necessary  it  may  be  done  by  simple  examination  of 
the  colonies  developed  on  the  plates  or  by  animal  inoculations  made 
from  the  water  used  in  the  air  filter.  It  is  sometimes  necessary  to  vary 
the  composition  of  the  medium  used  in  order  to  favor  the  development 
of  certain  types  of  organisms  desired,  for  example,  a  higher  precentage 
of  molds  will  be  found  and  a  more  luxuriant  development  will  take  place 
if  wort  agar  or  acid  gelatin  is  used. 

NUMBER  OF  BACTERIA  IN  THE  AIR. — The  number  of  bacteria  in  the 
air  is  determined  by  a  variety  of  conditions.  The  velocity  of  air  cur- 
rents and  the  nature  of  the  surface  with  which  these  currents  will  come 
into  contact,  are  probably  most  important.  Bacteria  are  usually  more 
abundant  on  quiet  days  in  the  air  of  buildings  than  out  of  doors,  but  on 
windy  days  the  reverse  is  true.  They  are  often  more  abundant  in  cities 
than  in  the  country.  Fewer  are  found  at  high  altitudes  and  over  large 
bodies  of  water.  Frankland  found  that  there  are  fewer  in  winter  than 
in  summer.  They  are  washed  from  the  air  during  rains.  Bright  sun- 
light destroys  many.  The  nature  of  the  soil  and  the  vegetation  cover- 
ing it  has  a  marked  influence.  The  following  figures  from  various 


THE   MICROORGANISMS    OF   THF   AIR 


307 


authors  are  appended  to  serve  as  an  index  to  what  may  be  expected  in 
the  air  content  of  bacteria. 


Locality 

Number  of  organisms 
per  cubic  meter 

Observer 

Outdoor  air  Boston  

100—150  bacteria. 

Sedgwick  and  Tucker. 

Open  air.            

50—  75  molds. 
100—150  bacteria. 

Fischer. 

Open  field  

250 

Uffelman. 

Seacoast  

IOO 

Uffelman. 

Mountain  altitude  200  meters 

o 

Pasteur. 

Mont  Blanc  

4—  ii 

Ellis 

Spitzbergen  (Arctic  Regions) 

o 

Levin. 

Middle  of  Paris  

4,000 

Ellis. 

Paris  Street.                          

7  .COO 

Fischer. 

Tailor's  Room  in  Whitechapel  

17,000 

Ellis 

Boot  Workshoo.  . 

2<?,000 

Ellis. 

SPECIES  OF  ORGANISMS  IN  THE  AIR. — Penicillium  is  the  most  com- 
mon mold  isolated  from  the  air.  Next  in  importance  are  Mucor, 
Rhizopus,  and  Aspergillus  in  the  order  given.  In  addition  to  these  a 
considerable  number  of  species  of  hyphomycetous  molds  are  occasion- 
ally found.  Torula,  but  not  true  yeasts,  are  usually  common.  Bac- 
teria are  either  spore-bearing  soil  bacilli  or  cocci.  Of  the  former,  B.  sub- 
tilis,  B.  mycoides,  and  related  forms  are  ubiquitous.  Sarcina  lutea  and 
Sarcina  aurantiaca  and  certain  other  chromogenic  cocci  are  to  be  found 
in  almost  every  plate  exposed.  Since  the  air  does  not  have  a  true  flora, 
the  species  as  well  as  the  number  of  bacteria  present  must  depend  en- 
tirely upon  the  character  of  the  environment. 


CHAPTER  II 

MICROBIAL  AIR  INFLUENCE  IN  FERMENTATION, 
DISEASES,  ETC. 

AIR  AS  A  CARRIER  OF  CONTAGION. — There  are  many  popular  mis- 
conceptions of  the  influence  of  air  upon  health.  Experience  early 
taught  that  exposure  to  the  night  air  in  certain  localities  or  to  swamp 
air  during  certain  seasons  was  generally  followed  by  disease.  Natur- 
ally, the  air  itself  was  held  responsible.  We  know  now  that  certain 
fevers,  malaria,  etc.,  are  caused  in  every  instance  by  infection  with 
specific  microorganisms  and  that  these  organisms  are  not  usually  car- 
ried by  the  air  but  by  insects,  such  as  the  mosquito,  in  water  and  food. 
Nor  can  the  emanations  from  decaying  organic  matter  or  sewer  gas  itself 
be  held  to  produce  disease  directly.  Before  the  establishment  of  the 
germ  theory  of  disease,  leading  sanitarians  held  that  sickness  was 
induced  by  the  gases  from  the  decaying  organic  matter,  by  the  effluvia 
from  cesspools  and  by  sewer  gas.  However  important  the  places  named 
may  be  in  harboring  disease  microorganisms,  we  have  learned  that  the 
air  itself  rarely  acts  as  a  carrier.  Sewer  gas  has  been  shown  to  be  un- 
usually free  from  bacteria.  Hazen  says,  "After  many  years  of  exper- 
ience and  long-continued  investigation,  there  is  not  the  slightest  reason 
to  believe  that  infectious  diseases  are  carried  by  the  air  of  sewers." 

Undoubtedly  the  air  does  play  some  part  in  the  carrying  of  disease 
germs.  In  certain  diseases,  as  the  exanthemata  (smallpox,  measles, 
etc.),  the  infecting  agent  may  be  present  on  the  dry  skin  and  may  be 
blown  about  and  inhaled.  This  means,  however,  is  not  established. 
In  certain  nasal,  tracheal,  and  pulmonary  infections,  the  organisms 
may  be  spread  through  speaking,  sneezing,  and  coughing,  for  the  infec- 
tious droplets,  as  has  been  seen,  remain  suspended  for  a  time  in  the 
air.  Pyogenic  cocci  are  present  in  the  mouth  and  care  must  be  used  in 
surgical  operations  that  the  mouth  is  so  protected  that  none  of  these 
organisms  gain  entrance  to  wounds.  Rarefy,  if  ever,  are  intestinal 
infections,  as  typhoid  or  cholera,  spread  through  the  air.  We  may  there- 
fore conclude  that  air  is  of  secondary  importance  as  a  carrier  of  infection. 

308 


MICROBIAL   AIR  INFLUENCE  309 

It  may  be  of  importance  in  a  crowded  workroom,  but  even  under  these 
conditions  it  is  probable  that  transmission  of  infection  comes  about 
more  frequently  through  actual  contact  or  through  food  and  drink. 

ORGANISMS  or  THE  AIR  AND  FERMENTATIONS. — A  uniform  inocula- 
tion with  soil  bacteria  such  as  produce  the  nodules  on  the  roots  of  leg- 
umes is  obtained  over  considerable  areas  through  the  action  of  the  wind 
in  blowing  dust  particles.  The  bacterial  flora  of  milk  is  to  some  extent 
dependent  upon  air  currents  as  is  also  the  development  of  the  molds 
necessary  to  the  proper  ripening  of  cheese,  such  as  the  Camembert. 
Acetic,  butyric,  and  other  organisms  are  likewise  distributed  in  this 
manner.  The  organisms  responsible  for  putrefaction  and  decay,  the 
molding  and  spoiling  of  foods  are  wind-borne. 

FREEING  AIR  FROM  BACTERIA. — Air  is.  most  commonly  freed  from 
bacteria  by  sedimentation,  for  this  is  the  ultimate  fate  of  most  dust  par- 
ticles. We  have  seen  that  they  gradually  subside  in  a  quiet  atmos- 
phere. When  large  quantities  of  pure  air  are  required,  dust  and  bac- 
teria may  be  removed  by  passage  through  a  spray  of  water  or  through 
various  types  of  niters,  such  as  cotton,  glass,  wool,  etc.  A  familiar 
example  of  this  type  of  filtration  is  the  laboratory  use  of  cotton  plugs  in 
test-tubes.  It  is  sometimes  necessary  to  resort  to  fumigation  to  destroy 
the  organisms  of  the  air  when  an  undesirable  species  is  present. 


DIVISION  II 

MICROBIOLOGY  OF  WATER  AND  SEWAGE 

CHAPTER  I* 

MICROORGANISMS  IN  WATERf 

Water  is  necessary  in  the  life  of  man.  Besides  its  use  as  a  beverage, 
for  cooking,  and  all  domestic  purposes,  it  is  largely  used  in  many  manu- 
facturing industries;  therefore,  the  study  of  its  chemical  and  biological 
content  is  one  of  the  most  important  features  of  modern  hygiene.  All 
natural  waters  contain  microorganisms,  which  gain  entrance  from  many 
sources. 

Under  the  influence  of  the  sun,  sea  water  evaporates  and  forms  a 
water  vapor,  which  we  call  clouds;  and  these,  driven  by  the  wind  over 
the  land,  are  precipitated  as  rain  and  in  the  form  of  snow  or  hail. 

Most  of  this  water  collects  from  vast  areas  into  brooks,  creeks, 
rivers,  lakes,  or  in  subterranean  streams,  and  finally  reaches  the  sea 
whence  it  came. 

The  water  vapor  arising  from  the  sea  or  land  contains  no  organisms ; 
but  as  soon  as  the  vapor  is  precipitated  microorganisms  find  their  way 
into  it.  These  come  from  the  air  and  from  the  soil.  Some  of  them  find 
in  water  sufficient  nutriment  for  their  life  and  growth;  and,  because  of 
their  constant  presence  and  evident  ability  to  thrive  in  water,  they  are 
sometimes  spoken  of  as  belonging  to  the  "wafer  flora"  Others,  such  as 

*  Prepared  by  F.  C.  Harrison. 

fFor  specific  details  regarding  methods  of  analysis  and  a  fuller  presentation  of  the  subject, 
readers  may  consult  any  of  the  following  excellent  books: 

1.  Savage,  W.  G.:  The  Bacteriological  Examination  of  Water  Supplies,  London,  H.   K. 
Lewis,  1906. 

2.  Horrocks,  W.  H.:  An  Introduction  to  the  Bacteriological  Examination  of  Water,  London, 
J.  and  H.  Churchill,  1901. 

3.  Prescott  and  Winslow:  Elements  of  Water  Bacteriology,  26.  Ed.,  New   York,  Wiley  & 
Sons,  1913. 

310 


MICROORGANISMS   IN   WATER  311 

the  soil  bacteria,  are  found  only  at  certain  seasons,  as  after  rain  or  dur- 
ing flood- time,  and  flourish  only  for  a  time;  while  some  few,  such  as 
intestinal  organisms  that  find  their  way  into  water,  survive  for  only 
a  short  period. 

CLASSES  OF  BACTERIA  FOUND  IN  WATER 

The  bacteria  found  in  water  are  here  roughly  divided  into:  (a)  natu- 
ral water  bacteria;  (b)  soil  bacteria  from  surface  washings;  (c)  intes- 
tinal bacteria,  usually  of  sewage  origin.  But  there  is  no  strict  divid- 
ing line  between  these  three  groups;  for  some  organisms  belonging  to 
the  water  flora  are  found  in  the  soil,  and  vice  versa.  Water  draining 
from  manured  land  frequently  contains  intestinal  organisms.  The 
division,  however,  is  sufficient  for  all  practical  purposes. 

NATURAL  WATER  BACTERIA. — The  natural  water  bacteria  are  gen- 
erally regarded  as  harmless  to  man.  These  organisms  are  frequently 
numerous  in  river,  lake,  and  all  surface  waters;  certain  species  predomi- 
nate at  one  season,  and  disappear  at  another.  Some  of  the  best  known 
are  mentioned  below.  Several  investigators  have  grouped  the  bacteria 
found  in  water  into  classes  according  to  their  biochemical  properties. 
Where  groups  are  subsequently  referred  to,  the  classification  is  that 
used  by  Jordan  and  followed  by  many  other  workers. 

B.  fluorescens  liquefaciens,  Group  V,  together  with  some  closely  allied 
varieties,  is  probably  more  frequently  found  in  water  than  any  other 
form,  and  is  easily  recognized  by  the  green  fluorescence  and  liquefaction 
it  produces  in  gelatin. 

B.  fluorescens  non-liquefaciens,  Group  VI,  as  the  name  implies,  does 
not  liquefy  gelatin,  but  produces  characteristic  colonies  with  a  fluores- 
cent shimmer,  is  often  very  abundant  in  river  waters,  and  is  representa- 
tive of  a  group  comprising  B.  f.  longus,  B.  f.  tennis.  B.  f.  aureus,  and 
B.  f.  crassus. 

Certain  organisms  which  liquefy  gelatin  and  acidify  milk — classed  by 
Jordan  in  his  Group  VIII — are  quite  common  at  certain  seasons. 
Some  of  these  are  soil  organisms  and  are  closely  related  to  the  proteus 
group;  and  some  of  them  are  B.  liquefaciens,  B.  punctatus,  B.  circulans. 

Chromo genie  bacilli  and  cocci  (Groups  XIII,  and  XIV)  are  often 
present  in  water.  Of  those  producing  red  coloring  matter,  the  well- 
known  B.  prodigiosus  is  the  type  of  the  group;  others  are  B.  ruber,  B. 


312  MICROBIOLOGY   OF   WATER. AND    SEWAGE 

indicus,  B.  rubescens  and  B.  rubefaciens.  Several  yellow  and  orange 
organisms  are  commonly  found,  such  as  B.  aquatilis,  B.  ochraceus,  B. 
aurantiacus,  B.  fulvus,  etc. 

At  certain  times,  particularly  in  river  and  brook  waters,  organisms 
producing  violet  pigment  are  quite  common.  B.  molaceus  or  B.  janthi- 
nus,  as  it  is  sometimes  called,  is  the  prevailing  type;  others  are  B.  limdus, 
B.  amethystinus,  and  B.  coeruleus. 

The  chromogenic  cocci  produce  either  orange  or  yellow  pigment,  and 
as  a  rule  are  not  numerous  in  water.  Sarcina  lutea  is  the  most  common 
species. 

Non-chromogenic  cocci  (Group  XV)  are  more  frequent.  M.  candi- 
cans,  M.  nivalis,  M.  aquatilis,  are  non-liquefying  forms,  and  M.  corona- 
tus  is  the  type  of  those  which  liquefy  gelatin. 

SOIL  BACTERIA  FROM  SURFACE  WASHINGS. — During  times  of  flood, 
high  water,  and  after  rains,  numerous  soil  organisms  are  found  in 
natural  waters;  and  occasionally  certain  species  persist  for  a  consider- 
able time.  Among  the  commonest  species  is  B.  mycoides,  with  its 
characteristic  rhizoid  colony;  also  B.  subtilis,  B.  megatherium,  and  B. 
mesentericus  vulgatus,  with  its  allied  varieties;  likewise  B.  m.fuscus  and 
B.  m.  ruber —  all  belonging  to  Jordan's  Group  VII,  and  having  many 
characters  in  common,  such  as  characteristic  colonies,  followed  by 
liquefaction  when  growing  in  gelatin,  production  of  spores,  etc. 

^Cladothrix  dichotoma,  one  of  the  thread  bacteria,  easily  recognized 
on  gelatin  plates  by  the  brown  halo  that  surrounds  the  colony,  is  often 
found  in  fresh  and  stagnant  water,  and  in  most  soils.  It  seems  to 
flourish  wherever  there  is  much  organic  matter. 

These  are  the  soil  organisms  most  often  found  when  beef  peptone 
gelatin  is  used  for  isolating  purposes;  but  if  other  media  are  used,  a 
different  flora  appears,  and  we  find  nitrifying  organisms,  yellow 
chromogens,  etc. 

INTESTINAL  BACTERIA,  USUALLY  OF  SEWAGE  ORIGIN. — Proteus 
Group. — There  are  several  groups  of  sewage  organisms  found  in  impure 
water;  some  of  these  are  very  abundant  in  crude  sewage,  but  are  not 
found  in  such  relatively  large  numbers  in  contaminated  water.  Jor- 
dan's Group  III  contains  the  organisms  belonging  to  the  large  proteus 
group,  the  principal  species  being  B.  vulgaris,  B.  zenkeri,  B.  mirabilis, 
B.  zopfii,  the  sewage  proteus  of  Houston,  and  B.  cloaca.  All  these  are 
frequently  found  in  impure  water,  and  in  sewage.  In  the  latter  Hous- 


MICROORGANISMS   IN   WATER  313 

ton  has  found  as  many  as  100,000  per  c.c.  All  these  organisms  are  mo- 
tile, liquefy  gelatin,  and  produce  gas  in  dextrose  and  saccharose  broth, 
and  little  or  none  in  lactose;  reduce  nitrates,  curdle  milk,  produce  indol, 
and  give  a  fecal,  disagreeable  odor  in  broth  or  other  media. 

Sewage  streptococci. — The  streptococci  found  in  sewage  are  probably 
similar  to  those  found  elsewhere;  but  their  appearance  in  contaminated 
water  may  be  regarded  as  indicative  of  recent  sewage  contamination, 
because  the  bulk  of  the  evidence  available  seems  to  show  that  they  are 
delicate  organisms,  which  rapidly  die  outside  of  the  body.  While  it  is 
easy  to  ascertain  their  presence  in  polluted  water,  it  is  almost  impossible 
to  enumerate  them;  and  they  do  not  furnish  such  good  evidence  of  sew- 
age pollution  as  the  colon  bacillus.  They  may  be  said  to  furnish  valu- 
able confirmatory  evidence  of  sewage  contamination. 

B.  enteritidis  sporo genes. — This  resistant,  spore-bearing  organism  is 
usually  present  in  the  intestinal  tract  of  man;  is  found  in  sewage,  milk, 
and  dust;  and  occurs  in  foodstuffs,  such  as  wheat,  oatmeal,  rice,  etc. 
On  account  of  its  ubiquity  and  the  resistance  of  its  spores,  it  cannot  be 
considered  a  good  indicator  of  excretal  pollution. 

B.  coli. — The  presence  of  this  organism  in  potable  water  is  gener- 
ally accepted  as  the  best  bacterial  indicator  of  sewage  pollution.  It 
must  be  remembered,  however,  that  there  are  many  varieties  of  this 
organism,  to  which  certain  investigators  have  given  specific  names,  even 
when  the  differences  from  the  type  organism  have  been  very  slight.  It 
may  be  well  to  mention  some  of  these,  to  avoid  confusion  in  the  mind  of 
the  reader.  The  true  colon  bacillus,  B.  coli,  or  B.  coli  communis,  or  B. 
coli  communis  verus,  is  a  short  bacillus  with  rounded  ends,  motile,  forms 
no  spores  and  is  Gram  negative,  does  not  liquefy  gelatin,  produces 
acidity  and  coagulation  in  litmus  milk,  gives  rise  to  acid  and  gas  in 
glucose  and  lactose  media,  causes  canary-yellow  fluorescence  in  neutral 
red  media,  and  produces  indol  when  grown  in  peptone  water.  The  term 
11  Excretal  B.  coli"  has  been  suggested  as  a  convenient  designation  of  an 
organism  which  possesses  the  above  characteristics. 

A  saccharose  fermenting  variety  of  B.  coli  has  been  named  B.  com- 
munior;  and  we  have  a  whole  series  of  organisms  which  differ  more  or  less 
in  various  biochemical  reactions,  or  lack  some  of  their  positive  reactions. 
To  some  of  these  the  name  "para-colon"  has  been  given;  and  the  name 
"para  typhoid"  has  been  applied  to  those  which  more  closely  approxi- 
mate to  the  cultural  peculiarities  of  the  typhoid  bacillus. 


MICROBIOLOGY   OF   WATER  AND    SEWAGE 


For  practical  purposes  in  the  analysis  of  water,  these  distinctions  are 
unnecessary. 

Bad.  lactis  aerogenes,  a  short,  thick,  capsulated,  non-motile 
bacterium  related  to  B.  coli,  is  also  an  intestinal  organism,  and  must  be 
regarded  as  an  indicator  of  sewage  pollution. 

B.  typhosus. — Very  few  instances  are  recorded  in  bacteriological 
literature  of  the  direct  isolation  of  the  typhoid  bacillus  from  infected 
water.  The  organism  is  not  long-lived,  even  in  pure  water  (eight 
or  ten  days);  and  when  exposed  to  the  action  of  sewage  bacteria,  its 
longevity  is  greatly  diminished  (not  more  than  five  to  six  days).  A 
few  resistant  specimens  may  remain  alive  for  longer  periods  of  time. 

Although  the  typhoid  bacillus  has  been  found  so  infrequently  in 
water,  it  is  well  understood  at  the  present  time  that  the  purification  of 
the  water  supply  of  a  town  or  city  produces  a  marked  decrease  in  the 
number  of  cases  and  in  the  mortality  from  typhoid  fever,  as  the  following 
table  shows:  (See  also  Fig.  122.) 

DEATHS  FROM  TYPHOID  FEVER  PER  100,000  PER  YEAR 


Place 

Purification 
by 

Date  of 
change 

Five  years 
before 
change 

Five  years 
after  change 

Percentage 
of 
reduction 

Filtration 

1802—  3 

47 

85 

Zurich 

Filtration 

i88«c 

76 

IO 

87 

Filtration 

180-? 

121 

26 

70 

Albany  N.  Y. 

Filtration 

1800 

IO4 

28 

77 

Not  only  has  such  a  marked  improvement  followed  the  purification 
of  public  water  supplies  in  the  case  of  typhoid  fever,  but  it  has  been 
shown  by  statistics  that  "where  one  death  from  typhoid  fever  has  been 
avoided  by  the  use  of  better  water,  a  certain  number  of  deaths,  probably 
two  or  three,  from  other  causes  have  been  avoided." 

In  the  routine  examination  of  water,  no  particular  effort  is  made  to 
isolate  this  organism,  owing  to  the  difficulty  of  the  task.  The  tests  that 
the  present-day  investigator  has  to  satisfy  are  extremely  thorough;  and 
unless  the  suspected  organism  conforms  to  the  whole  of  these  necessary 
tests  it  cannot  be  accepted  as  true  B.  typhosus. 

Msp.  comma. — The  spirillum,  or  vibrio,  of  Asiatic  cholera  is 
an  intestinal  organism;  and  the  disease  it  produces  is  spread  largely 
by  water.  Epidemics  of  cholera  are  more  easily  traced  to  their 


MICROORGANISMS    IN    WATER 


315 


AVERAGE  ANNUAL  DEATH  RATE  FROM  TYPHOID  FEVER  PER  100.000  OF  THE  POPULATION 
1912  I          10         20        30       -40       SO       60 


MUNICH 


OUN  'AIM 


VIENNA 

BERLIN 

ZURICH 

HAMBURG 

PARIS 

LONDON 

CLEVELAND.O. 

PATERSON.NJ. 

WATERTOWN.N.Y. 

CINCINNATI^. 

SEATTLE.WASH. 

CH1CAGO,ILL. 

ST.LOUIS.HO. 

MINNEAPOUS.HINN. 

PHILADELPHIAJPA. 

PITTSBURGH. PA. 

NEW  ORLEANS.LA. 

NEWYORK.N.Y. 

SPRINGFIELD.MASS. 

BINGHAMPTON,N.Y. 

ALBANY,  NY. 

LAWRENCE.MASS. 

RICHMOND.VA. 

BALTIMORE.MD. 

HILWAUKEE,WIS. 

TOLEDO.O. 

ATLANTA. 

BIRHINGHAH.ALA. 

WHEEUNG.W.VA. 

MEMPHIS/TENN. 

ATLANTA,GA. 


SP 


LTEF 

EUROF 


UNG 


/ATERS 

CIT  ES 


An  instructive  contrast  between  Altona  and  Hamburg  before  the  latter  filtered 
its  water,  having  learnt  its  lesson  from  a  sharp  outbreak  of  cholera. 


A  FEW 

SCATTERED 


CASES  OP  CHOLERA. 
ALTOHA 


HAMBURG. 

POPULATION:  600.000 
CHOLERA  CASES:  17.000 

-       DEATHS:    8.60O 


ALTONA: 


HAMBURG: 
W< 


WATER  FILTERED 

!U 
ATER  UM FILTERED 


FIG.  122.— (After  G.  E.  Armstrong.) 


3i6 


MICROBIOLOGY   OF   WATER   AND    SEWAGE 


source  than  those  of  typhoid  fever,  owing  to  the  "explosive"  character 
.of  the  disease.  At  the  time  of  the  outbreak  of  cholera  in  Hamburg,  in 
1892,  the  cholera  vibrios  were  frequently  isolated  from  the  water  of  the 
river  Elbe,  which  was  used  to  furnish  the  regular  supply  of  the  city. 
The  adjoining  city  of  Altona  also  obtained  its  water  from  the  same 
river,  after  it  had  received  some  of  the  Hamburg  sewage;  yet  it  remained 
practically  free  from  the  scourge,  owing  to  the  efficiency  of  sand  filters 
which  were  used  to  purify  the  water  (Fig.  122).  In  times  of  epidemic, 
the  organism  has  been  isolated  from  rivers,  wells,  and  reservoirs  in 
India,  a  country  in  which  the  disease  is  endemic. 


THE  NUMBER  OF  BACTERIA  IN  RAIN,  SNOW,  HAIL,  ETC.,  AND  IN  WATER 
FROM  WELLS,  UPLAND  SURFACE  WATERS,  RIVERS,  AND  LAKES 

RAIN. — The  number  of  bacteria  found  in  rain  depends  upon  the 
month  of  the  year  and  the  dryness  of  the  air.  When  considerable  dust 
is  present  in  the  air,  the  first  rain  beats  it  back  to  the  soil;  and  at 
such  time  rain  water  contains  more  organisms  than  usual.  Rain  falling 
in  densely  inhabited  cities  always  contains  more  microbes  than  rain 
falling  on  open  farm  land  or  upland  pastures.  A  few  figures  will  be 
sufficient  to  illustrate. 

NUMBER  OF   BACTERIA  PER  LITER  OF  RAIN   WATER 
Figures  for  Montsouris  Park,  Paris,  France,  and  the  average  for  two  years 


Month 

Number  of  organisms 
per  liter 

Month 

Number  of  organisms 
per  liter 

January  

8,OOO 

July.. 

"\,6oo 

February 

I  32O 

August 

8  2.00 

March    

2.02O 

September  

5,770 

April 

2  I4.O 

October 

2,220 

May  

2,44.0 

November      

3,2^0 

June. 

e  6oO 

December 

4.,  7  5Q 

Yearly  average  5,300  per  liter  per  month. 


The  average  for  the  interior  of  Paris  corresponds  with  the  larger 
amount  of  dust  in  the  air,  and  reaches  a  total  of  19,000  organisms  per  L. 
With  a  yearly  rainfall  of  609.6  mm.  (24  inches),  the  rain  washes 
down  during  the  year  some  5,000,000  organisms  to  the  square  yard. 


MICROORGANISMS    IN   WATER  317 

SNOW. — The  results  obtained  from  snow  are  similar  to  those  ob- 
tained from  rain;  but  as  a  rule  the  numbers  are  larger,  a  result  doubtless 
due  to  the  larger  particles  of  the  snow  flakes.  One  investigator  has 
found  from  334  to  463  bacteria  per  c.c.  of  snow  water.  On  the  sum- 
mit of  high  mountains  snow  is  practically  sterile,  Binot  not  finding 
a  single  organism  in  8  c.c.  of  water  from  mountain-top  snow. 

Water  issuing  from  glaciers  is  of  remarkable  purity,  containing 
only  from  three  to  eight  organisms  per  c.c.;  but  the  numbers  are  larger 
as  the  distance  from  the  glacier  increases. 

HAIL. — Hail  stones  usually  contain  large  numbers  of  bacteria, 
varying  from  628  to  21,000  per  c.c.  of  water  obtained  from  the  melt- 
ing hail.  Fluorescing  bacteria  have  been  found  in  some  samples; 
and  the  presence  of  these  microorganisms  suggests  that  surface  water 
is  sometimes,  carried  up  by  storms  and  congealed.  The  presence  of 
many  molds  in  hail  is  due  to  contamination  from  the  air. 

DEEP  WELLS. — Deep  well  water  and  spring  water  contain  as  a 
rule  but  few  organisms,  usually  less  than  50  per  c.c.  on  gelatin  at  20°, 
and  less  than  5  per  c.c.  on  agar  plates  at  blood  heat.  In  a  series  of 
tests  of  water  taken  direct  from  forty-three  artesian  wells,  152.4  M. 
(500  feet)  deep  or  more,  the  writer  has  found  an  average  of  27  per 
c.c.  for  the  gelatin  and  1.5  per  c.c.  for  the  agar  counts.  These  tests 
have  extended  over  a  period  of  several  years;  and  water  from  deep 
springs  has  given  similar  results. 

SHALLOW  WELLS. — The  bacterial  content  of  shallow  wells  depends 
greatly  on  their  location  and  construction.  Even  in  those  well  lo- 
cated and  constructed,  the  number  varies  with  the  amount  of  rainfall, 
and  is  often  large.  In  polluted  wells,  very  high  numbers  of  organisms 
are  found. 

Sedgwick  and  Prescott  found  from  190  to  8,640  bacteria  per  c.c. 
in  unpolluted  wells. 

In  the  same  class  of  wells,  Savage  found  from  10  to  100  per  c.c.  by 
the  blood-heat  count,  and  100  to  20,000  or  more  by  the  gelatin  count. 

Sixty  polluted  wells  examined  by  the  writer  gave  an  average 
gelatin  count  of  740  bacteria  per  c.c.;  and  thirty-eight  wells  which  were 
free  of  contamination  gave  an  average  count  of  400  per  c.c. 

Polluted  wells  often  give  counts  approximating  the  higher  numbers 
mentioned  above;  but,  of  course,  the  character  of  the  bacterial  flora 
is  quite  different. 


3l8  MICROBIOLOGY   OF   WATER  AND    SEWAGE 

UPLAND  SURFACE  WATERS. — There  are  few  bacteria  in  upland  sur- 
face waters  draining  barren  uplands.  Cultivation,  grazing  of  animals, 
and  human  habitation  produce  other  conditions.  In  pure  waters, 
50  to  300  per  c.c.  by  the  gelatin  and  i  to  10  by  the  agar  count  are  found. 

RIVERS. — The  greatest  variation  in  the  number  of  bacteria  exists 
in  river  waters.  Many  factors,  such  as  sewage  contamination,  tempera- 
ture, rain  fall,  vegetable  debris,  etc.,  influence  the  microbial  popu- 
lation. A  few  figures  may  be  given  for  illustration. 

BACTERIOLOGICAL  EXAMINATION  OF  RIVERS  AT  AND  BELOW  LARGE  SOURCES  OF 
POLLUTION  (BOYCE  AND  CO-WORKERS) 


Distance 

Direction 

Munich. 
River  Isar 

Cologne. 
River  Rhine 

About    0.6  mile  

Above 
Below 

305 
0,^87 

4,786 

About    2  7  miles  . 

Below 

la.fJO^ 

About    6.0  miles  

Below 

8,764 

30,4^2 

About  12  o  miles  . 

Below 

4,7o6 

I2,4.6O 

Below 

3,6o2 

O.CQtr 

About  26  o  miles  . 

Below 

7,860 

In  the  Chicago  drainage  canal,  Jordan  found  1,245,000  bacteria 
per  c.c.  at  Bridgeport;  650,000  at  Lockport,  twenty-nine  miles  below; 
and  3,660  at  Averyville,  159  miles  below.  Below  where  the  sewage  of 
Peoria  enters,  the  number  rises  to  758,000  at  Wesley  City,  and  decreases 
to  4,800  at  Kampsville,  123  miles  from  Peoria. 

The  River  Rhone  contains  an  average  of  75  bacteria  per  c.c.  above 
Lyons  and  800  below.  The  Dee,  88  above  Braemar  and  2,829  Per  c-c- 
below.  Many  more  similar  results  are  found  in  the  literature. 

LAKES. — The  water  of  lakes  is  generally  much  purer  than  river 
water.  Near  the  shore,  the  bacterial  content  is  higher  than  farther 
out,  showing  the  contaminating  influence  of  habitation.  Thus  Lake 
Geneva  contains  as  many  as  150,000  bacteria  per  c.c.  near  the  shore, 
and  further  out  only  38  per  c.c.  Other  figures  are  as  follows:  Loch 
Katrine,  74  per  c.c.,  Lake  Lucerne,  8  to  51  per  c.c.,  Lake  Champlain, 
82  per  c.c. 

SEA  WATER. — There  are  few  bacteria  in  sea  water  remote  from 
the  coast;  but  near  the  shore  and  in  the  neighborhood  of  seaports 
there  may  be  large  numbers. 


MICROORGANISMS   IN   WATER  319 

Examples:  350  M.  from  Naples,  sea  water  contained  26,000  bac- 
teria per  c.c.  At  a  distance  of  3  KM.,  only  10.  Samples  taken  from 
depths  of  75  to  800  M.  at  distances  from  4  to  15  KM.  from  shore  were 
found  to  contain  from  6  to  78  bacteria  per  c.c.  in  surface  water,  and 
from  3 'to  260  at  various  depths  below. 

CAUSES  AFFECTING  THE  INCREASE  AND  DECREASE  OF  THE 
NUMBER  OF  BACTERIA  IN  WATER 

There  is  a  number  of  causes  which  influence  the  multiplication 
or  diminution  of  microorganisms  in  natural  waters;  and  while  it  is 
necessary  to  discuss  each  of  these  causes  in  detail,  it  must  be  remem- 
bered that  a  number  of  them  may  be  simultaneously  influencing  the 
increase  or  decrease. 

TEMPERATURE. — In  natural  waters,  a  low  temperature  probably 
acts  injuriously  on  parasitic  bacteria,  reducing  their  numbers;  but 
the  bacterial  content  of  water  during  the  hot  summer  months  is  gener- 
ally not  so  large  as  during  the  cooler  seasons.  Water  collected  for 
examination  should  be  analyzed  at  once;  otherwise,  contradictory 
results  as  to  numbers  will  be  found.  Usually,  in  most  waters,  there  is 
a  reduction  in  numbers  for  a  few  hours,  followed  by  a  large  increase. 
Very  much  polluted  waters,  however,  show  a  marked  decrease  of 
intestinal  organisms,  if  the  samples  are  kept  cool. 

LIGHT. — Although  the  germicidal  effect  of  sunlight  is  well  known, 
yet  it  has  not  such  powerful  effects  on  the  bacteria  in  water. 
Much  depends,  no  doubt,  on  the  turbidity  and  speed  of  the  cur- 
rent, the  maximum  killing  effect  being  produced  in  shallow,  clear 
and  slow-moving  water.  It  has  been  found  by  experiment  that  the 
germ-killing  power  of  light  extends  to  a  depth  of  3  M  (about  9.84  feet). 
As  a  means  of  purifying  water,  direct  light  produces  very  little  effect. 

FOOD  SUPPLY. — The  amount  of  organic  matter  in  water  directly 
influences  the  growth  of  bacteria.  Where  a  large  amount  of  this  is 
present,  the  number  of  microorganisms  is  also  large.  Rivers  containing 
considerable  organic  matter  derived  from  vegetable  debris,  etc.,  contain, 
as  a  rule,  more  organisms  than  rivers  in  which  there  is  but  little  of 
such  material.  Thus  the  Ottawa  River,  which  drains  a  large  area  of 
forest  lands  and  is  characterized  as  an  upland  peaty  water  carrying  a 
rather  high  percentage  of  organic  and  volatile  matter,  contains  through- 


320  MICROBIOLOGY    OF    WATER   AND    SEWAGE 

out  the  year  a  larger  number  of  organisms  to  the  cubic  centimeter 
than  the  water  of  the  river  St.  Lawrence,  which  is  much  clearer  and 
contains  much  less  organic  matter.  Sewage  water  is  rich  in  organic 
matter,  and  proportionately  rich  in  bacterial  life;  and  bacterial  purifica- 
tion is  synchronous  with  a  diminution  of  organic  matter. 

Jordan  remarks  in  this  connection  that  "in  the  causes  connected 
with  the  insufficiency  or  unsuitability  of  the  food  supply  is  to  be  found 
the  main  reason  for  the  bacterial  self-purification  of  streams." 

OXIDATION. — On  the  surface  of  waters,  in  rapids,  falls,  and  tidal 
rivers,  much  oxygen  is  absorbed,  and  much  impure  matter  is  oxidized. 
Such  oxidation  is  one  of  the  minor  agencies  in  the  purification  of  water. 

VEGETATION  AND  PROTOZOA.— Low  forms  of  plant  and  animal  life, 
like  certain  species  of  algae,  river  plants,  and  the  numerous  protozoan 
forms,  bring  about  a  reduction  of  organic  matter  in  water,  and  thus 
reduce  the  amount  of  food  available  for  bacteria.  There  is  also  the 
antagonism  between  these  forms  and  bacteria.  The  chemical  products 
of  the  higher  forms  are  considered  by  some  authorities  to  be  injurious 
to  bacterial  life;  and  many  bacteria  are  ingested  by  predatory  protozoa. 

DILUTION. — Sewage  flowing  into  a  river  or  lake  is  at  once  diluted 
with  quantities  of  pure  water,  and  the  amount  of  available  food  mate- 
rial is  thus  diminished;  the  space  occupied  by  a  definite  number  of  bac- 
teria is  increased;  and  it  is  easy  to  see  that  the  greater  the  dilution, 
the  fewer  sewage  bacteria  will  be  found.  An  example  will  suffice  to 
illustrate.  The  sewage  of  the  city  of  Ottawa  amounts  to  about 
454  L.  (100  gallons)  per  second;  and  the  gelatin  count  from  it  gives 
an  average  in  round  numbers  of  3,000,000  bacteria  per  c.c.  The 
yearly  mean  discharge  of  the  river  is  about  1,364,511  L.  (300,000 
gallons)  a  second;  and  thus  the  sewage  becomes  diluted  3,000  times. 

SEDIMENTATION. — Impurities,  suspended  matter,  and  bacteria 
having  weight,  naturally  gravitate  to  the  bottom;  and  the  subsidence 
of  these  matters  is  spoken  of  as  sedimentation. 

Lake  water  being  still,  sedimentation  in  it  is  more  marked  than  in 
moving  water;  and  such  water  contains  but  few  bacteria.  In  slow- 
moving  rivers  the  influence  of  this  factor  is  also  quite  pronounced; 
and,  according  to  Jordan,  "The  influences  summed  up  by  the  term 
sedimentation  are  sufficiently  powerful  to  obviate  the  necessity  for 
summoning  another  cause  to  explain  the  diminution  in  numbers 
of  bacteria"  in  sewage  polluted  rivers.  The  example  already  given 


MICROORGANISMS   IN   WATER  321 

of  the  self -purification  of  the  Chicago  drainage  canal  illustrates  Jordan's 
contention. 

OTHER  CAUSES. — There  is  a  number  of  other  causes,  not  well 
known  nor  of  sufficient  practical  importance  for  more  detailed  com- 
ment, which  may  increase  or  decrease  the  number  of  bacteria  in  water, 
such  as  the  inhibiting  action  of  microorganisms  and  their  products 
on  one  another,  the  effects  of  pressure,  etc. 

A  peculiar  fact,  which  has  never  been  satisfactorily  explained,  is  the 
quick  death  (in  three  to  five  hours)  of  the  cholera  vibrio  in  the  waters 
of  the  Ganges  and  Jumna.  When  one  remembers  that  these  rivers 
are  grossly  contaminated  by  sewage,  by  numerous  corpses  of  natives 
(often  dead  of  cholera),  and  by  the  bathing  of  thousands  of  natives,  it 
seems  remarkable  that  the  belief  of  the  Hindoos,  that  the  water  of  these 
rivers  is  pure  and  cannot  be  defiled,  and  they  can  safely  drink  it  and 
bathe  in  it,  should  be  confirmed  by  means  of  modern  bacteriological 
research.  It  is  also  a  curious  fact  that  the  bactericidal  power  of 
Jumna  water  is  lost  when  it  is  boiled;  and  that  the  cholera  vibrio 
propagates  at  once,  if  placed  in  water  taken  from  wells  in  the  vicinity 
of  the  rivers. 

INTERPRETATION  OF  THE  BACTERIOLOGICAL  ANALYSIS  OF  WATER 

In  making  any  analysis  of  water,  all  data,  such  as  the  kind  of 
water  and  the  particulars  regarding  collection,  transmission,  sampling, 
rainfall,  etc.,  should  be  given,  as  these  are  a  great  help  in  interpreting 
the  results.  One  analysis  is  rarely  sufficient;  examinations  should 
be  regularly  and  systematically  made. 

QUANTITATIVE  STANDARDS. — No  absolute  guide  can  be  given  to 
determine  the  potable  quality  of  water  from  the  number  of  micro- 
organisms in  it.  It  may,  however,  be  safely  assumed  that  high  bacte- 
rial counts  indicate  a  large  amount  of  organic  matter.  The  number  of 
organisms  growing  in  beef  peptone  gelatin  at  20°  to  22°,  and  termed 
the  "gelatin  count,"  should  be  given.  For  deep  wells  and  springs, 
this  should  not  exceed  50  per  c.c.;  and  for  shallow  wells  and  rivers, 
not  over  500  per  c.c.  After  rains  or  floods,  these  figures  might  be 
exceeded,  and  would  not  necessarily  indicate  dangerous  pollution. 

The  number  of  organisms  which  develop  on  beef  peptone  agar 
incubated  at  blood  heat,  commonly  termed  the  "agar"  or  "blood- 
si 


322  MICROBIOLOGY   OF    WATER   AND    SEWAGE 

heat"  count,  is  perhaps  more  important  than  the  gelatin  count,  as 
many  water  bacteria  do  not  grow  at  blood  heat,  whereas  sewage  and 
soil  organisms  grow  readily  at  this  temperature.  The  agar  count 
eliminates  the  water  flora,  but  obscures  the  sanitary  results  by  reason 
of  the  presence  of  soil  bacteria.  For  deep  waters,  the  agar  count 
should  generally  not  exceed  10  per  c.c.;  and  for  surface  waters,  not 
over  100  per  c.c. 

QUALITATIVE  STANDARDS. — The  isolation  and  identification  of 
specific  disease  organisms,  such  as  typhoid  and  cholera  microbes 
from  water,  is  sufficient  to  condemn  such  a  sample  as  unfit  for  use; 
but,  on  account  of  many  technical  difficulties,  it  is  practically  impossible 
to  make  such  an  examination.  Apart  from  a  few  special  cases,  when 
it  may  be  necessary  to  attempt  the  isolation  of  these  pathogenic 
bacteria,  the  presence  of  the  colon  bacillus  (B.  coli)  in  small  amounts 
of  water,  is  generally  looked  upon  as  significant  and  indicative  of  sew- 
age pollution.  The  technical  methods  used  in  this  isolation  and 
enumeration  are  many,  and  may  be  found  in  the  works  cited;  but  there 
is  considerable  difference  of  opinion  as  to  the  number  of  B.  coli  which 
should  condemn  a  sample  of  water.  Prescott  and  Winslow  state 
that  if  the  colon  bacillus  is  in  "such  abundance  as  to  be  isolated  in  a 
large  proportion  of  cases  from  i  c.c.  of  water,  it  is  reasonable  proof 
of  the  presence  of  serious  pollution."  Savage  suggests  that  B.  coli 
should  be  absent  from  100  c.c.  in  the  case  of  water  from  deep  wells 
and  springs,  and  should  be  absent  from  10  c.c.  in  surface  waters,  such 
as  rivers  used  for  drinking  purposes,  shallow  wells,  and  upland  surface 
waters. 

The  streptococcus  examination  is  next  in  importance  as  an  indi- 
cator of  sewage.  Streptococci  should  be  absent  from  the  amounts 
of  water  mentioned  above  for  B.  coli;  and  B.  enteritidis  sporogenes 
should  not  be  present  in  1,000  c.c.  of  water  from  deep  wells,  nor  in 
100  c.c.  from  surface  waters. 

SEDIMENTATION,  FILTRATION,  AND  PURIFICATION  OF  WATER 

As  areas  become  more  and  more  thickly  settled  and  towns  and 
cities  increase  in  population,  the  problem  of  obtaining  sanitary  con- 
trol over  the  water  supply  increases  in  importance.  Very  few  towns 
and  cities  are  fortunate  to  obtain  their  water  supply  from  an  unpol- 


MICROORGANISMS    IN    WATER 


323 


luted  area.  Consequently  expensive  installation  must  be  made,  in  order 
to  purify  a  suspiciously  contaminated  water  by  freeing  it  from  organ- 
isms injurious  to  health.  There  are  several  methods  of  accomplish- 
ing such  purification;  and  these  will  be  briefly  mentioned. 

SEDIMENTATION  AND  FILTRATION. — This  method  of  purifying  water 
has  been  used  for  nearly  a  hundred  years;  but  the  great  impetus  given  to 
this  hygienic  measure  was  due  to  Koch,  who  showed  in  1893  that  the 


FIG.  123. — Section  of  a  sand  filter. 

proper  nitration  of  Elbe  water  saved  the  town  of  Altona  from  an  epi- 
demic of  cholera  which  devastated  Hamburg  as  a  result  of  drinking  un- 
filtered  water.  In  this  system  of  purification,  the  water  is  first  stored  in 
large  reservoirs,  where  the  effect  of  sedimentation  and  storage  reduces 
considerably  the  number  of  bacteria.  From  the  reservoir,  the  water  is 
filtered  through  sand,  gravel,  and  pebbles,  etc.,  arranged  as  shown 
in  Fig.  123.  This  filtration  removes  from  97  to  99.5  per  cent  of  the 
microorganisms. 

The  action  of  the  filter  bed  is  due  to  the  mechanical  obstruction  of 
impurities,  to  oxidation  of  the  organic  matter,  and  to  ni truncation  due 


324  MICROBIOLOGY    OF    WATER   AND    SEWAGE 

MEAN  OF  MONTHLY   EXAMINATIONS  FOR  THE   YEAR 


MicroSrganisms  per  c.c. 

At  source 

After  storage 

After  filtration 

London,  Lambeth  Works 

16,138 
16,138 
1,400 
79,000 
186,986 

7,820 
1,067 

75 
34 
60 
630 
400 

London,  Chelsea  Works  

Berlin,  Lake  Muggel  

Paris,  Marne  

Paris,  Seine  

to  the  living  bacteria  in  the  scum  which  forms  on  the  top  of  the  layer  of 
sand.  Of  these,  the  last  is  the  most  important;  for  until  this  gelatinous 
layer  forms,  the  filter  does  not  act  properly— in  fact,  it  has  little  filter- 
ing action,  as  the  following  figures  show: 

BACTERIAL  CONTENT  OF  WATER  BEFORE  AND  AFTER  CLEANING  THE  SAND  FILTER 

Before  cleaning,  i.e.,  before  removing  the  scum  layer. . .  42  per  c.c. 

One  day  after  cleaning 1880 

Two  days  after  cleaning 752 

Three  days  after  cleaning 208 

Four  days  after  cleaning 156 

Five  days  after  cleaning 102 

Six  days  after  cleaning 84 

Thus  provision  must  be  made  to  permit  the  scum  or  film  to  form  be- 
fore the  filtered  water  is  used  for  domestic  purposes. 

The  rate  of  filtration  must  be  regulated;  for  if  the  water  is  allowed  to 
exceed  a  certain  rate  (101.6  mm.  or  4  inches  per  hour),  inefficiency 
follows. 

COAGULATING  BASINS  AND  FILTRATION. — This  method  of  purifica- 
tion consists  in  adding  a  coagulant,  such  as  basic  sulphate  of  aluminum, 
by  means  of  a  mechanical  device  which  regulates  the  quantity,  as  the 
water  is  pumped  into  the  coagulating  basins  or  reservoirs,  where  it  re- 
mains for  six  to  twenty-four  hours.  The  aluminum  sulphate  is  decom- 
by  the  lime  in  the  water  and  forms  insoluble  aluminum  hydrate; 
the  sulphuric  acid  combines  with  the  lime.  The  hydrate  of  alumi- 
num is  precipitated  in  large  flocculent  masses,  entangling  all  particles 
of  soil  or  organic  matter;  and  these,  being  deposited  on  the  surface  of  the 


MICROORGANISMS   IN   WATER 


325 


sand,  form  the  filtering  layer.     Such  filters  are  very  efficient;  they  re- 
move from  97  to  99.8  per  cent  of  the  bacteria  from  the  water. 

POROUS  FILTERS. — (Fig.  124.)  These  filters  are  made  either  from 
unglazed  porcelain  or  baked  diatomaceous  earth;  the  former  are  known 
as  Chamberland,  and  the  latter  as  Berkefeld  filters.  These  filters 
are  usually  candle-shaped,  require  considerable  pressure  to  force  water 
through  them,  and  can  be  used  only  when  a  small  supply  of  water 
is  needed.  Water  which  is  forced  through  these  filters  is  at  first  sterile; 
but  with  repeated  use  they  allow  bacteria  to  pass  through  the  pores  and 


FIG.  124. — Unglazed  porcelain  niters.     Chamberland  system;  A,  without  pressure; 
B,  fitted  to  main  water  supply;  C,  section  of  a  porous  porcelain  filter. 

thus  the  filtering  efficiency  is  impaired  and  will  remain  so,  until  the  fil- 
ters are  cleaned  and  baked  to  red  heat  in  a  muffle-furnace.  Unless  this 
is  done  regularly,  no  dependence  should  be  placed  on  these  filters,  as 
they  only  put  those  who  use  them  off  their  guard  against  the  danger  to 
which  they  are  exposed. 

PURIFICATION  BY  OZONE. — The  antiseptic  properties  of  ozone  are 
well  known.  It  is  used  in  the  purification  of  the  water  supply  of  some 
towns — Nice,  Chartres,  etc.  Ozone  used  for  this  purpose  is  usually 
obtained  by  means  of  the  electric  current;  and  a  flowing  film  of  water  is 


326  MICROBIOLOGY   OF   WATER   AND    SEWAGE 

brought  into  contact  with  an  upward  current  of  air  charged  with  ozone, 
which  current  makes  the  water  almost  completely  sterile.  This  method 
of  purification  is  efficient,  but  rather  expensive. 

PURIFICATION  BY  HEAT. — By  bringing  water  to  the  boiling  point,  all 
harmful  bacteria  are  destroyed;  a  few  spores  may  resist  this  treatment, 
but  they  are  harmless.  Boiled  water  is  of  a  flat,  insipid  taste,  due  to  the 
driving  out  of  the  contained  gases.  The  taste  may  be  improved  by 
cooling  and  shaking.  The  boiling  of  water  is  often  resorted  to  as  a  hy- 
gienic measure  in  times  of  epidemic,  and  for  the  supply  of  armies  in  the 
field. 

PURIFICATION  BY  CHEMICALS. — The  addition  of  a  small  amount  of 
calcium  hypochlorite,  or  potassium  iodide,  etc.,  purifies  water;  but  these 
methods  are  seldom  used,  except  for  the  use  of  soldiers  on  campaign. 
Hypochlorite,  however,  is  now  used  more  commonly  in  municipal  water 
supplies  where  they  can  not  be  otherwise  controlled. 

LOCATION  AND  CONSTRUCTION  OF  WELLS 

Farms  in  many  sections  of  this  country  are  practically  all  supplied 
with  surface  water  collected  in  shallow  wells.  Hence  farmers  should 
understand  the  principles  involved  in  the  location  and  construction 
of  wells. 

Many  farm  wells  are  badly  located — too  near  such  sources  of  con- 
tamination as  outhouses,  cesspools,  stables,  or  barnyards;  and  those 
who  locate  them  give  too  little  attention  to  the  slope  of  the  ground,  and 
the  nature  and  slope  of  the  subsoil.  There  should  be  at  least  22  to 
30  M.  (75  to  100  feet)  between  the  well  and  all  probable  sources  of 
contamination;  and  this  distance  is  too  small,  if  the  soil  is  very  porous, 
or  if  the  surface  and  subsoil  drainage  is  toward  the  well,  or  if  the  well 
is  sunk  in  fissured  rock — as  it  is  obvious  that  there  are  serious  chances 
of  contamination  in  each  of  the  above  circumstances. 

In  all  cases,  the  surface  drainage  should  be  away  from  the  well;  and, 
as  far  as  possible,  the  subsoil  drainage  also  should  be  from  the  well. 

Sketches  125,  126,  and  127  illustrate  these  points,  the  upper  part  of 
each  drawing  showing  the  plan  and  the  lower  portion  a  section  through 
the  dotted  line  marked  on  the  plan.  Fig.  125,  shows  that  the  surface 
drainage  is  from  the  house,  privy,  stables,  and  barnyard  to  ward  the  well. 
The  section  through  the  line  "A"  shows  the  relation  of  the  impervious 


MICROORGANISMS   IN    WATER 
[Dm  m/ 


327 


FIG.  125. 


< 

. 

I-1. 

R  /  s^ 

j 

/ 


A 


FIG.  126. 


FIG.  127. 

FIGS.  1 25, 1 26,  and  127. — In  each  figure — plan  above — section  through  A  B  below. 
S  =  soil;  B  =  impervious  subsoil  or  strata,  r,  House;  2,  well;  3,  outhouse;  4, 
piggery;  5,  stables;  6,  stable  yard;  7,  hen  house;  8,  sheep  stable.  Arrow  heads 
indicate  direction  of  water  flow.  (Original.) 


328 


MICROBIOLOGY   OF   WATER   AND    SEWAGE 


subsoil  "  B  "  to  the  drainage.  Water  falling  on  the  surface  of  the  ground 
would  penetrate  through  the  soil  to  the  upper  portion  of  the  subsoil,  and 
then  move  along  it  in  the  direction  of  the  greatest  slope.  In  this  sketch, 
the  subsoil  drainage  is  away  from  the  well;  and  in  this  respect  the  well  is 
located  properly;  but,  in  respect  to  the  surface  drainage,  improperly 
located.  A  better  place  for  the  well  would  be  at  the  letter  "X". 

In  Fig.  126  the  surface  drainage — including  that  from  the  adja- 
cent outhouse  at  3,  which  is  too  close  to  the  well — is  toward  the  barn, 
and  away  from  the  well;  but  the  subsoil  drainage  from  all  the  buildings, 


Soli 


FIG.  128. — Construction  of  a  model  well.     On  the  right  is  brick  construction,  on 
the  left  stone  construction,  as  illustrated.     (Original.) 

except  the  house,  is  in  the  direction  of  the  well;  and  thus  contamination 
of  the  water  supply  is  liable  to  occur. 

Fig.  127  shows  a  well  properly  located  as  regards  both  surface  and 
subsoil  drainage.  Such  a  well  will  supply  pure  water,  if  it  is  properly 
constructed. 

Fig.  128  shows  the  proper  construction  of  a  well  with  brick  or  stone. 
Large  vitrified  drain  pipes  with  cemented  joints  will  answer  equally  well 
when  there  is  an  abundant  supply  of  water;  but  in  case  the  supply  of 


MICROORGANISMS    IN   WATER  329 

water  is  limited,  a  large  area  is  needed,  and  a  stone  or  brick  well  is 
necessary. 

Reference  to  the  illustrations  will  show  that  every  endeavor  is  made 
to  prevent  surface  water  from  entering  directly  into  the  well.  The  walls 
are  impervious;  and  the  earth  or  clay  is  well  rammed  against  the  outer 
side  of  the  wall.  The  curb  is  carried  well  above  the  surface  of  the 
ground.  The  waste  water  is  conducted  by  means  of  a  sloping  platform, 
trap,  and  drain,  away  from  the  well;  and  the  well  opening  is  properly 
covered.  All  water  entering  such  a  well  must  percolate  through  a  con- 
siderable depth  of  soil,  and  undergo  purification  by  means  of  the  aggre- 
gations of  living  bacteria  in  the  soil  spaces.  Thus  the  soil  around  a  well 
fulfils  the  same  function  in  purifying  the  surface  water  as  the  scum 
layer  that  forms  on  the  surface  of  gravel  filters. 


CHAPTER  II* 

MICROBIOLOGY   OF   SEWAGE 
THE  BACTERIAL  FLORA  OF  SEWAGE 

COMPLEXITY  OF  FLORA. — Sewage  is  made  up  of  the  miscellaneous 
and  varied  wastes  of  human  life  and  activity,  and  the  bacteria  which  are 
found  therein  are  the  result  of  a  haphazard  and  chance  admixture  of 
substances  of  diverse  origin  and  character.  The  resulting  flora  is  not 
only  of  great  diversity  and  variability,  but  it  is  with  few  exceptions  non- 
characteristic.  In  brief,  the  medium  with  which  we  have  to  deal  has 
had  an  origin  too  indefinite  and  a  history  too  short  to  have  permitted 
the  establishment  of  anything  approaching  a  constant  or  characteristic 
bacterial  flora. 

TYPICAL  FORMS. — Our  interest  in  this  sewage  flora  is  a  very  practical 
one,  being  confined  to  those  organisms  which  carry  on  the  work  of  bio- 
logical purification  and  to  certain  pathogens  which  for  obvious  reasons 
require  special  treatment.  We  are  interested  chiefly  in  what  these  bac- 
teria do  rather  than  in  what  they  are,  and  our  classification  is  influenced 
accordingly.  It  is  based,  not  upon  the  species  or  the  genus  nor  even 
upon  the  group  or  type,  that  proves  so  convenient  in  general  bacterial 
classification,  but  upon  a  sort  of  physiological  or  functional  type,  having 
to  do  solely  with  the  activities  of  the  organisms  in  sewage  and  in  its  puri- 
fication. Bacteria  performing  a  common  function  or  producing  a  com- 
mon result  are  members  of  one  type.  Individuals  may  belong  to  several 
of  our  types  and  there  are  doubtless  a  great  many  that  belong  to  none. 
These  latter  simply  have  no  place  assigned  them  as  yet  in  the  role  of 
sewage  purification,  because  they  possess  none  of  the  recognized  typical 
functions. 

Apparent  exception  may  be  taken  to  these  general  principles  in 
the  case  of  such  organisms  as  the  B.  coli,  sewage  streptococci  and 
B.  enteritidis.  These  are,  to  a  certain  extent,  characteristic  sewage 
bacteria.  But  interest  hi  them  as  individuals  is  confined  to  water 

•Prepared  by  Earle  B.  Phelps. 

330 


MICROBIOLOGY   OF   SEWAGE  331 

bacteriology.  If  they  have  any  functions  in  the  bacterial  changes  of 
sewage,  they  receive  attention  as  members  of  a  corresponding  type,  not 
as  individuals.  A  study  of  these  sewage  types,  therefore,  is  a  study  of 
the  chemical  changes  induced  in  the  medium  by  the  activities  of  one  or 
the  other  group  of  bacteria. 


TYPES  OF  SEWAGE   BACTERIA 

According  to  the  general  character  of  the  changes  which  they  bring 
about,  sewage  bacteria  are  divided  into  two  large  groups,  the  anaerobic 
or  putrefactive  bacteria,  and  the  oxidizing  bacteria.  In  regard  to  the 
former,  no  attention  is  paid  to  the  fine  distinctions  that  have  been  made 
in  recent  years  in  connection  with  the  definition  of  putrefaction.  In 
sewage  chemistry  putrefaction  is  that  change  which  takes  place  natur- 
ally in  sewage  after  anaerobic  conditions  have  become  established.  It 
involves  the  reduction  of  urea,  the  hydrolysis  of  protein  and  of  cellulose, 
the  emulsification  of  fats,  the  reduction  of  nitrates  and  sulphates  and 
possibly  of  phosphates,  and  those  other  changes  which  are  characterized 
by  the  withdrawal  of  oxygen  and  the  hydrolysis  of  complex  molecules. 
These  changes  are  always  noted  in  sewage  under  anaerobic  conditions 
and  the  terms  putrefactive  and  anaerobic  change  are  for  the  present  pur- 
poses practically  synonymous. 

The  oxidizing  reactions  on  the  other  hand  might  be  classed  under 
the  general  heading  of  aerobic  reactions,  except  that  they  constitute 
only  a  small  portion  of  the  group  of  reactions  which  take  place  normally 
under  aerobic  conditions.  They  are  distinguished  by  the  fact  that  oxy- 
gen is  added  to  the  molecule,  the  product  always  containing  more  oxy- 
gen than  the  initial  substance.  Carbon  dioxide,  water  and  nitrates  are 
produced,  in  distinction  from  methane,  hydrogen  and  ammonia,  which 
characterize  the  anaerobic  reactions.  A  third  type,  possessing  objec- 
tive rather  than  subjective  functions,  in  sewage,  is  made  up  of  patho- 
genic and  other  harmful  bacteria.  These  play  no  part  in  our  theories 
of  purification  and  the  proof  of  their  presence  is  generally  lacking. 
For  the  protection  of  the  public  health,  it  is  assumed  that  they  are 
always  present  in  sewage,  and  our  procedure  in  sewage  disposal  is  modi- 
fied throughout  in  accordance  with  this  assumption. 

With  these  definitions  in  mind  we  may  proceed  to  a  more  detailed 
study  of  the  bacterial  types  themselves. 


332  MICROBIOLOGY   OF    WATER   AND    SEWAGE 

PUTREFACTIVE  AND  ANAEROBIC  BACTERIA. — Putrefaction  or  anae- 
robic fermentation  involves  the  withdrawal  of  oxygen  from  one  molecule 
or  part  of  a  molecule  and  the  subsequent  oxidation  of  another  molecule 
or  part  of  the  same  molecule.  The  energy  released  in  this  process  is 
utilized  in  the  vital  functions  of  the  organism.  This  action  is  neither 
oxidation  nor  reduction,  or  more  strictly,  they  are  both  taking  place 
simultaneously. 

A  good  example  of  such  a  process  is  the  fermentation  of  urea.  The 
reaction  takes  place  as  follows: 

CO(NH2)2  +  2H2O  =  (NH4)2CO3. 

Carbon  is  oxidized  at  the  expense  of  hydrogen,  a  process  which,  by  itself, 
is  endothermic,  that  is,  requires  heat  or  energy  for  its  maintenance. 
But  the  heat  of  formation  of  the  final  product  is  greater  than  that  of  the 
initial  substances  and  the  energy  thus  liberated  becomes  available  for 
use  by  the  bacteria.  It  is  in  this  way  that  hydrolytic  changes  of 
this  character  play  the  same  r61e  in  anaerobic  reactions  that  is  played 
by  direct  oxidation  under  aerobic  conditions. 

The  Liquefaction  of  Protein. — One  of  the  most  clearly  defined  and 
useful  types  of  bacterial  activity  to  be  seen  in  the  various  sewage 
disposal  processes  is  that  which  we  term  liquefaction.  This  term  is 
used  to  denote  broadly  all  those  changes  by  which  solid  and  insoluble 
organic  matter  is  converted  into  a  soluble  condition.  The  particular 
process  known  as  protein  liquefaction  is  in  the  main  analogous  to  gas- 
tric digestion.  Its  one  characteristic  is  the  increased  solubility  of  the 
product.  The  practical  importance  of  protein  liquefaction  in  sewage 
disposal  is  very  great  and  the  value  of  the  liquefying  bacteria  corre- 
spondingly high.  Nevertheless,  aside  from  our  knowledge  of  analogous 
processes  in  digestion  and  in  bacterial  putrefaction  of  albuminous  sub- 
stances, we  know  almost  nothing  of  the  chemistry  or  the  bacteriology 
of  this  process.  An  enormous  variety  of  bacteria  are  included  in  this 
group.  The  whole  process  is  doubtless  the  result  of  a  very  complicated 
symbiosis  in  which  various  sub-groups  of  bacteria  carry  out  the  initial  re- 
action, from  which  point  other  groups  carry  it  through  successive  stages. 
Absence  of  one  or  another  of  these  groups  or  of  some  important  species  of 
any  group  doubtless  accounts  for  the  diverse  results  that  are  recorded. 
It  is  well  known  that  the  activities  within  a  septic  tank,  for  example, 


MICROBIOLOGY   OF   SEWAGE  333 

are  seldom  twice  the  same.  Gross  differences  readily  apparent  to  the 
senses  of  one  versed  in  such  matters  certainly  exist,  and  in  actual  results 
it  is  rare  to  find  two  tanks  doing  exactly  the  same  kind  of  work.  Much 
depends  of  course  upon  the  chemical  character  of  the  sewage  itself,  but 
much,  that  is  still  unexplained,  must  eventually  be  traced  to  the  great 
diversity  of  the  sewage  flora  and  the  complex  symbiosis  as  well  as  bac- 
terial antagonisms  that  are  involved  in  the  reactions  with  which  we 
are  dealing. 

During  these  reactions  proteins  and  albumins  are  hydrolyzed  by  suc- 
cessive stages  to  albumoses,  peptones,  amino-acids,  amines,  and  finally 
to  ammonia,  carbon  dioxide,  methane,  hydrogen,  etc.  Simultaneously 
ammonia,  amines,  and  carbon  dioxide  are  eliminated  at  each  stage  as 
products.  The  tendency  then  is  toward  simple,  soluble  and  gas- 
eous side  products,  and  hence  of  value  in  the  preliminary  resolution 
of  the  sewage. 

The  Fermentation  of  Cellulose. — The  fermentation  of  cellulose  is, 
next  ,to  protein  hydrolysis,  the  most  important  work  of  the  anaerobic 
bacteria  in  sewage  treatment.  So  far  as  is  definitely  known  this  action 
is  usually  confined  to  anaerobic  conditions.  The  fact  that  fence  posts 
decay  first  at  the  surface  of  the  ground,  or  that  wood  in  general  decays 
more  rapidly  when  it  is  exposed  to  only  a  slight  degree  of  moisture,  than 
when  it  is  immersed  in  water  is  only  an  apparent  contradiction.  The 
conditions  are  aerobic  in  both  cases  and  aerobic  bacteria  would  not  be 
favored  by  total  immersion  but  the  effect  in  both  instances  seems  to  be 
due  to  fungus  growths  which  are  more  active  in  the  moist  wood. 

The  anaerobic  fermentation  of  cellulose  is  that  which  is  found  typ- 
ically in  marshes  and  of  which  the  chief  products  are  carbon  dioxide  and 
methane  or  "marsh  gas."  Nitrogenous  food  material  is  also  requisite, 
which  accounts  for  the  preserving  property  of  reasonably  pure  water 
upon  wood. 

In  the  septic  tank  the  solution  of  cellulose  is  extremely  rapid,  and 
large  pieces  of  cotton  cloth  or  rolls  of  paper  are  completely  dissolved 
within  a  few  months.  Wood  itself  is  more  resistant  and  withstands  the 
action  of  the  tank  for  years.  This  is  largely  due  to  the  fact  that  the 
wood  molecule  is  much  more  complicated  than  a  simple  cellulose 
molecule,  and,  among  the  conifers  at  least,  to  the  further  fact  that 
antiseptic  intercellular  substances  are  present. 

Chemically  considered  the  action  is  hydrolytic  and  can  be  imitated 


334  MICROBIOLOGY   OF   WATER   AND    SEWAGE 

by  prolonged  boiling  in  dilute  acids.  Pectin  substances,  starches  and 
finally  sugars  are  produced  while  butyric  and  other  organic  acids,  carbon 
dioxide  and  methane  appear  as  by-products.  Bacteriologically,  al- 
though it  has  variously  been  ascribed  to  one  or  another  organism,  it  is 
probably  the  result  of  the  activities  of  many  and  is  possibly  not  the 
principal  activity  of  any  one  of  these.  In  other  words,  cellulose  fermen- 
tation is  probably  a  series  of  side  reactions  produced  during  the  fermen- 
tation of  the  nitrogenous  material  rather  than  a  definite  reaction  upon 
which  the  metabolism  of  any  single  species  depends.  This  view  is 
strengthened  by  the  general  observations  that  this  fermentation  is  in 
most  cases  due  directly  to  enzymes.  Viewed  in  this  light  it  is  easy  to 
understand  the  difficulty  that  has  surrounded  the  isolation  of  definite 
cellulose  fermenting  organisms.  Many  have  been  described,  chief  of 
which  are  B.  butyricus  or  B.  amylobacter,  B.  omelianski,  Sp.  rugula. 

The  Saponification  of  Fats. — A  third  great  group  of  type  reactions 
occurring  under  anaerobic  conditions  is  the  saponification  or  split- 
ting of  fat.  Our  knowledge  of  this  process  is  even  less  definite 
than  of  the  cellulose  fermentations.  It  is  a  fact  that  there  does  take 
place  in  sewage  a  gradual  saponification  and  emulsification  by  which 
the  fat  loses  its  identity  and  mingles  with  the  liquid.  This  effect  is 
most  noticeable  in  the  case  of  long  sewers  in  which  considerable  veloci- 
ties are  maintained.  In  quiescent  tanks  there  is  a  tendency  for  the  fats 
to  rise  to  the  surface  and  thus  become  removed  from  the  influence  of 
this  action.  Thus  in  small  installations  enormously  heavy  scums  form 
upon  the  tanks  and  analysis  shows  a  considerable  percentage  of  fat 
in  this  material.  In  larger  systems  on  the  other  hand  there  is  less  and 
less  evidence  of  fatty  material  as  such.  It  is  true  that  there  is  a  deposit 
upon  the  walls  and  tops  of  such  sewers  and  that  small  floating  objects, 
like  matches,  rolling  along  such  a  wall  will  accumulate  layers  of  grease 
and  become  eventually  the  familiar  "grease-balls"  found  in  the  dis- 
charge, but  in  the  main  the  fatty  material  has  become  well  disintegrated 
before  the  outlet  is  reached. 

In  this  case  also  as  in  that  previously  discussed  it  is  not  believed  that 
the  action  is  a  direct  result  of  the  activity  of  any  particular  organism. 
The  proteolytic  changes  are  accompanied  by  the  freeing  of  alkaline 
products,  ammonia  and  amines,  which  leads  to  some  saponification. 
and  which,  in  turn,  leads  to  a  further  emulsification.  It  has  also  been 
demonstrated  that  bacterial  activity  is  commonly  associated  with  fat 


MICROBIOLOGY   OF    SEWAGE  335 

saponification  and  decomposition.  Whether  specific  enzymes  are  pres- 
ent which  assist  in  this  final  process  or  not  has  never  been  determined. 
It  is  significant  to  note,  however,  that  where  sewages  are  slightly  acid, 
unaltered  fats  are  much  more  abundant,  even  though  the  acidity  is 
insufficient  to  prevent  vigorous  putrefactive  changes  in  the  sewage 
itself. 

The  Fermentation  o]  Urea. — The  fermentation  of  urea  has  already 
been  referred  to  as  a  typical  and  simple  case  of  anaerobic  decomposition. 
This  reaction  has  great  significance  in  sewage  chemistry  since  a  consider- 
able proportion  of  the  nitrogen  of  sewage  is  present  initially  as  urea. 
Owing  to  the  ease  and  rapidity  with  which  the  reaction  takes  place, 
however,  no  special  effort  is  necessary  to  bring  it  about  in  sewage 
treatment  and  it  therefore  receives  brief  attention  in  discussions  of  the 
chemistry  of  sewage.  -  The  change  to  ammonia  takes  place  in  the  small 
sewers  of  the  system  and  it  is  difficult  and  generally  impossible  to  detect 
the  presence  of  urea  in  sewage.  It  has  even  been  suggested  that  certain 
enzymes  present  in  fecal  matter  are  instrumental  in  bringing  about  this 
change  and  that  the  bacteria  are  only  indirectly  concerned.  It  is 
known,  however,  that  a  large  number  of  bacteria  of  general  occurrence 
have  the  power  to  produce  this  fermentation.  Of  these  the  Bact.  urea 
(Miquel)  may  be  cited  as  an  example. 

The  Reduction  of  Sulphates  and  Nitrates. — The  production  of  sul- 
phuretted hydrogen  during  the  anaerobic  decomposition  of  sewage 
is  commonly  noted.  This  substance  may  arise  in  at  least  two  ways. 
Sulphur,  being  a  constituent  of  most  protein  substances,  is  split  off 
from  the  molecule  in  this  form  during  certain  types  of  fermentation. 
Its  formation  in  these  cases  is  analogous  to  that  of  ammonia  from 
protein.  The  amount  so  produced  is  small  and  is  usually  neutral- 
ized and  precipitated  by  the  small  amounts  of  iron  and  other  metals 
always  present  in  sewage.  There  is  therefore  no  liberation  of  the 
gas  itself  and  it  is  often  said  that  sulphuretted  hydrogen  is  not  formed 
normally  in  a  septic  tank.  This  conclusion  is  readily  disproved  by 
a  simple  test  of  the  black  residue  found  at  the  bottom  of  such  tanks. 

A  second  and  more  important  source  of  this  substance  is  the  sul- 
phate normally  present  in  many  sewages.  Throughout  many  parts 
of  the  country  the  water  supply  contains  material  quantities  of  mag- 
nesium or  calcium  sulphate,  and  upon  the  sea  coast  the  sewage  gener- 
ally receives  more  or  less  salt  water. 


336  MICROBIOLOGY   OF    WATER   AND    SEWAGE 

In  these  cases  the  reduction  of  sulphates  to  sulphuretted  hydro- 
gen is  not  only  of  interest  'bacteriologically  but  probably  exerts  an 
influence  upon  all  the  reactions  that  are  going  on  simultaneously. 
In  fact  this  example  serves  excellently  to  illustrate  the  great  complex- 
ity of  these  anaerobic  reactions  and  the  mutual  interdependence  of 
each  upon  all  the  others.  Sulphates,  under  anaerobic  conditions, 
are  a  source  of  oxygen  and  it  is  upon  oxygen  that  the  course  of  all  these 
reactions  depends.  Therefore  the  presence  of  sulphates  and  the 
possibility  of  their  yielding  oxygen  may  alter  the  course  of  the  other 
reactions  involved.  The  products  of  the  protein  hydrolysis  for  ex- 
ample may  be  profoundly  modified  by  the  presence  of  this  additional 
source  of  oxygen. 

The  effect  upon  the  bacteria  themselves  is  also  to  be  considered 
as  a  factor  quite  distinct  from  the  purely  chemical  effect  just  de- 
scribed. It  has  frequently  been  observed,  and  in  fact  would  be  ex- 
pected, that  the  products  of  anaerobic  putrefaction  are  themselves 
detrimental  to  the  activity  of  the  organism  producing  the  changes 
in  question.  The  nature  of  sulphuretted  hydrogen  makes  it  appear 
quite  probable  that  we  are  dealing  here  with  a  toxic  substance  that 
would  at  least  inhibit  the  activities  of  certain  bacteria  and  in  this  way 
further  modify  the  final  result. 

The  same  might  be  said  of  almost  all  the  reactions  with  which  we 
have  to  deal  but  this  example  is  cited  as  a  typical  one. 

It  is  known  in  practice  that  the  presence  of  sulphates  in  a  sewage 
does  lead  to  a  distinct  type  of  anaerobic  change  which  is  characterized 
by  the  marked  blackening  of  the  sewage,  the  formation  of  secondary 
reaction  products  which  precipitate  after  the  removal  of  the  suspended 
matter  of  the  sewage,  the  evolution  of  hydrogen  sulphide,  an  excessive 
amount  of  mineral  or  non-volatile  residue  in  the  sludge  and  the  forma- 
tion of  free  sulphur  upon  subsequent  aeration  of  the  sewage. 

Here  again,  as  in  the  other  types  of  reaction,  it  is  useless  for  the  pres- 
ent to  attempt  to  ascribe  this  reaction  to  any  particular  species.  Sp. 
desulphuricans  and  B.  sulphur eus  have  been  isolated.  A  non-liquefy- 
ing anaerobic  bacillus,  which  reduced  sulphates  strongly,  was  isolated 
from  Boston  sewage  in  the  writer's  laboratory  by  G.  R.  Spaulding. 
Others  have  been  described  and  there  is  undoubtedly  a  large  group  of 
organisms  capable  of  bringing  about  the  reaction. 

Just  as  the  reduction  of  nitrates  is  a  function  performed  by  many, 


MICROBIOLOGY   OF    SEWAGE  337 

perhaps  most,  anaerobes,  so  the  reduction  of  sulphates,  although  a 
less  common  function,  is  still  common  to  many  forms.  In  fact  ni- 
trates, sulphates,  and  phosphates  form  a  series  in  regard  to  their 
reducibility  and  the  effect  of  their  presence  upon  the  reaction  as  a 
whole.  The  phosphates  so  far  as  has  been  recorded  are  not  ordinarily 
reduced. 

OXIDIZING  BACTERIA.  The  Production  of  Nitrate  and  Nitrite. — A 
long  series  of  investigations  upon  the  organisms  which  oxidize 
nitrogen  began  with  the  Franklands  and  Winogradski,  and  has 
continued  to  the  present  day.  These  have  given  us  much  in- 
formation concerning  the  habits  and  functions  of  the  nitrifying 
organisms.  Winogradski's  original  types  were  Nitrosomonas  and 
Nitrobacter,  the  former  oxidizing  ammonia  to  nitrite,  the  latter 
completing  the  oxidation  to  nitrate.  Work  upon  these  organisms 
constitutes  such  .an  important  factor  in  soil  bacteriology  to-day 
that  more  detailed  discussion  of  this  nitrifying  function  is  left  for 
another  place. 

In  the  earlier  days  of  sewage  purification  great  stress  was  laid  upon 
the  work  of  these  organisms,  which  was  believed  to  be  fundamental. 
The  degree  of  nitrification  was  accepted  as  a  measure  of  the  work  of 
the  filters  and  little  thought  was  given  to  the  possibility  of  oxidizing 
reactions  by  other  forms.  With  the  development  of  modern  sewage 
disposal  methods,  the  work  of  this  latter  type  of  bacteria  has  assumed 
a  more  important  role  and  the  actual  work  of  the  nitrifying  organism 
has  been  found  to  be  of  only  minor  and  incidental  importance. 

Other  Oxidizing  Reactions. — The  great  groups  of  aerobic  and  facul- 
tative bacteria  are  in  general  concerned  in  the  oxidation  of  organic 
matter.  There  is  nothing  specific  in  this  reaction  and  very  little  that 
is  characteristic  of  any  special  or  smaller  groups.  Under  certain  special 
and  restricted  conditions,  typical  products  are  formed  by  particular 
species,  as  in  the  manufacture  of  vinegar,  and  it  is  possible  that  a  care- 
ful study  of  the  complex  reactions  involved  in  the  oxidation  of  sewage 
would  show  a  certain  sequence  in  the  order  of  events  and  certain  definite 
work  being  accomplished  by  definite  groups.  In  other  words,  symbio- 
sis and  specialization  doubtless  take  place  to  a  limited  extent.  But 
the  fundamental  fact  remains  that  the  metabolism  of  the  organism 
demands  that  organic  matter  be  oxidized  for  the  production  of  energy. 
Even  though  certain  food  substances  may  be  preferred  and  certain 

22 


33$  MICROBIOLOGY   OF   WATER   AND    SEWAGE 

decompositions  be  normally  produced  there  is  necessarily  a  great 
latitude  and  great  adaptability. 

For  this  very  reason  a  study  of  the  individual  organism  and  its 
action  upon  specific  materials  throws  no  light  upon  the  major 
problem,  which  is,  given  fifty  different  types  of  organisms  and  fifty 
different  fermentable  substances,  in  a  mixture,  what  will  be  the  course 
of  the  reaction?  Here  the  preferences,  the  adaptability  and  the  antag- 
onisms all  come  into  play  and  while  it  is  impossible  to  say  what  has 
happened  or  how,  it  is  readily  conceived  and,  in  fact,  almost  apparent, 
that  out  of  this  heterogeneous  mixture  there  will  come  a  homogeneous 
symbiotic  family  and  an  orderly  sequence  of  chemical  events,  in 
which  metabolic  needs  and  food  supply  are  all  delicately  adjusted. 

PATHOGENIC  BACTERIA.  Prevalence  and  Longevity. — Owing  to  its 
origin  and  nature,  sewage  may  at  any  time  contain  infectious  material 
and  for  the  purposes  of  the  sanitarian  it  is  assumed  that  at  all  times  the 
germs  of  disease  are  present.  Such  an  assumption  is  possibly  in  excess 
of  the  actual  facts  and  is  only  justified  because  it  supplies  the  only  pos- 
sible hypothesis  having  an  adequate  margin  of  safety.  The  actual 
prevalence  of  pathogenic  bacteria  obviously  depends  in  the  first  instance 
upon  the  amount  of  sickness  in  the  contributing  community.  Further- 
more, if,  as  we  are  coming  to  believe,  a  definite  proportion  of  the  popu- 
lation are  perpetual  carriers  of  typhoid  infection  then  to  just  as  definite 
an  extent  is  the  bacterial  population  of  the  sewage  made  up  of  typhoid 
bacteria  from  apparently  well  persons.  In  addition  to  these,  about 
five  one-hundredths  of  i  per  cent  of  the  population  of  American  cities 
are  suffering  from  the  disease  in  acute  form.  Making  due  allowance 
for  the  extra  precautions  that  are,  or  should  be  taken  in  the  care  of 
the  dejecta,  these  persons  constitute  a  definite  and  fairly  constant 
source  of  infection. 

In  the  case  of  the  other  infectious  diseases  of  the  alimentary  tract, 
and,  possibly  to  a  less  extent  in  the  case  of  tuberculosis,  diphtheria,  and 
many  others,  these  general  statements  are  equally  applicable,  so  that 
the  possibility  of  the  occurrence  of  infectious  material  in  sewage  is 
not  a  remote  one,  but  definite  and  almost  quantitatively  determinable. 

As  to  the  persistence  of  active  pathogenic  bacteria  in  the  sewage  for 
any  length  of  time  the  data  are  less  exact.  In  the  case  of  typhoid  fever, 
which  has  been  more  carefully  studied  than  any  other  disease,  the  germs 
are  more  persistent  in  pure  water  than  in  impure,  but  whether  this 


MICROBIOLOGY   OF   SEWAGE  339 

generality  can  be  extended  to  sewage  is  debatable.  Our  best  informa- 
tion leads  to  the  belief  that  any  reduction  in  numbers  of  typhoid 
bacteria  which  may  take  place  within  the  sewer  before  discharge  is  of 
minor  importance  and  of  slight  sanitary  significance. 

Discussion  of  other  pathogens  must  be  in  even  more  general  terms. 
Information  is  almost  wholly  lacking  and  it  can  only  be  assumed  for 
purposes  of  safety  that,  in  so  far  as  organisms  of  these  various  types  are 
discharged  into  the  sewer,  they  will  persist  to  a  certain  extent  in  the 
sewage  until  it  is  finally  disposed  of.  If  such  disposal  be  by  discharge 
into  a  stream  without  purification,  then  the  waters  of  that  stream 
become  polluted  with  infectious  material.  Studies  recently  made  by 
Sedgwick  and  McNutt  have  indicated  the  possibility  that  many  dis- 
eases, other  than  the  oft-quoted  typhoid  fever,  may  be  transmitted 
in  this  way. 

Life  in  Septic  Tanks  and  Filters. — With  the  introduction  of  the 
septic  tank  at  Exeter,  England,  in  1893,  the  question  of  the  fate  of 
pathogenic  bacteria  in  such  a  tank  was  raised.  It  was  even  suggested 
that  bacteria,  such  as  the  typhoid  organism,  might  multiply  in  the 
tank.  The  question  was  investigated  by  Professor  Sims  Woodhead, 
who  concluded  that  no  organisms  capable  of  setting  up  morbid  changes 
in  animals  were  discharged  from  the  tank.  This  negative  evidence, 
however,  has  little  weight  in  the  light  of  more  recent  experiments. 
Pickard  introduced  an  emulsion  of  typhoid  bacteria  into  this  same  tank 
and  noted  only  a  gradual  decrease.  After  fourteen  days  he  was  able 
to  detect  i  per  cent  of  the  initial  number.  He  also  reported  a  removal 
of  90  per  cent  of  the  typhoid  organisms  introduced  into  a  contact 
filter.  These  data  must  be  interpreted  in  the  light  of  two  established 
facts.  The  typhoid  organism  tends  to  die  at  a  rapid  but  diminishing 
rate  under  any  but  the  most  favorable  conditions.  This  results  in  a 
rapid  decrease  at  first,  with  a  prolonged  survival  of  a  few  individuals. 
This  process  takes  place  in  sewers,  in  streams,  and,  in  fact,  under  most 
artificial  conditions.  The  second  fact  of  importance  is  the  difficulty 
of  recovering  the  typhoid  organism  under  experimental  conditions  like 
those  described. 

A  thorough  study  of  the  bacteriology  of  sewage  and  of  filter  effluents 
led  Houston  to  conclude  that  the  biological  processes  at  work  in  a  filter 
or  tank  were  not  strongly  inimical,  if  hostile  at  all,  to  the  vitality  of 
pathogenic  germs. 


340  MICROBIOLOGY   OF   WATER  AND   SEWAGE 

A  conservative  study  of  all  the  evidence  bearing  upon  this  impor- 
tant question  including  the  vitality  and  fate  of  certain  non-pathogenic 
species,  such  as  B.  coli,  leads  to  the  conclusion  that  the  removal  of 
pathogenic  bacteria  in  purification  methods  is  due  to  two  allied  causes, 
the  efficiency  of  which  can  be  approximately  determined.  There  is 
first  the  time  element  and  the  known  rapid  decrease  in  the  numbers  of 
certain  bacteria  such  as  B.  typhosus  when  placed  under  conditions  that 
preclude  multiplication.  The  rate  of  decrease  varies  but  is  roughly 
about  50  per  cent  in  twenty-four  hours. 

The  second  factor,  acting  in  reality  in  conjunction  with  the  first, 
is  the  mechanical  hindrance  that  is  offered  to  the  free  passage  of  sus- 
pended materials  through  the  body  of  a  filter.  Even  fine  sand  offers 
little  straining  action  as  such,  since  the  open  channels  are  thousands 
of  times  as  big  as  the  bacterial  cell,  but  surface  tension  phenomena 
tend  to  make  all  solid  material  adhere  to  the  medium  and  thus  its 
passage  is  delayed.  This  action  is  prominent  although  of  less  impor- 
tance in  coarse-grained  filters.  Actual  experiments  by  the  writer  have 
indicated  that  while  the  liquid  may  pass  through  a  trickling  filter 
in  half  an  hour,  small  suspended  particles  such  as  ultramarine  and  B. 
prodigiosus  cells  require  an  average  of  over  twenty-four  hours.  In 
this  way  the  actual  time  of  passage  is  greatly  delayed  even  when  coarse 
broken  stone  is  the  filter  medium,  and  the  times  that  are  now  known 
to  be  necessary  for  the  passage  are  ample  in  themselves  to  account  for 
the  reductions  that  have  been  noted. 

It  may  therefore  be  stated  as  a  conservative  view  of  the  efficiency 
of  purification  processes  in  the  removal  of  pathogenic  bacteria,  that 
there  are  no  strongly  inimical  processes  at  work  in  the  tanks  or  filters, 
and  that  the  rate  of  decrease  is  not  materially  greater  than  would  be 
observed  in  the  same  period  of  time  under  the  conditions  of  a  running 
stream. 

THE  CULTIVATION  or  SEWAGE  BACTERIA 

There  are  two  general  methods  employed  for  the  cultivation  of 
those  bacteria  which  are  of  assistance  in  sewage  purification.  They 
may  be  cultivated  in  so-called  filters  of  sand  or  coarser  material,  or 
in  specially  constructed  tanks  such  as  the  septic  or  the  hydrolytic  tank. 
In  the  former  case  the  bacterial  growth  occurs  upon  the  special  medium 
provided,  the  sand  or  stone;  in  the  latter,  it  takes  place  in  the  liquid 


MICROBIOLOGY   OF   SEWAGE  341 

itself  and  a  continuous  life  history  within  such  a  tank  is  possible  only 
when  the  rate  of  flow  is  sufficiently  slow  to  permit  of  the  inoculation  of 
the  incoming  stream  by  the  contents  of  the  tank. 

FILTERS. — The  filtering  media  most  commonly  employed  are  sand 
or  crushed  stone  or  other  coarse  material.     In  natural  sand   beds  a 


FIG.  129. — Sewage  Experiment  Station,  Mass.  Inst.  Technology.  Trickling 
filter  in  front,  sand  filter  just  behind  filter,  dosing  tank  just  behind  sand  filter,  and 
septic  tank  just  behind  dosing  tank. 

brief  period  of  treatment  with  sewage  suffices  to  produce  an  active 
state  of  "nitrification."  By  this  term  is  indicated  alt  the  complex 
processes  of  oxidation  one  index  of  which  is  the  formation  of  nitrates. 
After  such  a  filter  has  once  become  active  in  this  way  it  will  continue, 
with  proper  care,  to  oxidize  sewage  almost  indefinitely.  Improper  care, 
such  as  an  overdose  of  sewage  or  continued  flooding  of  the  surface  due 
to  poor  drainage,  will  soon  destroy  the  activity  of  the  filter.  The  addi- 
tion of  germicidal  substances  has  a  similar  effect  and  cold  weather  some- 


342 


MICROBIOLOGY    OF    WATER   AND    SEWAGE 


what  reduces  the  efficiency.  From  all  this  it  is  apparent  that  a  filter 
is  a  biological  culture  medium  upon  which  the  various  types  of  bacteria 
are  growing  and  carrying  out  their  functions  and  that  such  a  medium 
requires  careful  control  and  is  sensitive  to  unfavorable  changes  in 
environment. 

The  other  filters  are  similar  to  this  and  illustrate  the  true  function  of 
filtration.  In  the  case  of  the  sand  filter  it  might  be  maintained  that 
filtration  or  straining  was  an  essential  element  in  the  process,  but  in  the 
case  of  these  coarse-grained  media  straining  action  is  eliminated.  Here 
there  is  nothing  but  a  pile  of  stones,  varying  from  i  to  3  inches  or 
more  in  diameter,  upon  the  surface  of  which  the  bacteria  grow.  The 


FIG.  130. — Sketch  of  septic  tank.     (Original.) 

sewage  trickles  slowly  over  the  surfaces,  or  is  held  in  contact  with  them 
temporarily,  according  as  we  are  dealing  with  trickling  or  contact  filters. 
Solids  adhere  to  the  stones  or  settle  upon  them,  and  soluble  material  is 
"absorbed "  by  the  surface  growth  and  removed  from  solution.  Within 
these  gelatinous  growths  to  which  the  air  also  has  free  access,  the  proc- 
esses of  oxidation  take  place  and  the  products,  the  semi-oxidized 
organic  material,  are  later  "shed"  from  the  stones  appearing  again  in 
the  effluent  as  humus  or  stable  organic  matter. 

ANAEROBIC  TANKS. — The  cultivation  of  bacteria  in  anaerobic  tanks 
is  not  quite  as  simple  a  matter  as  that  which  has  just  been  described. 


MICROBIOLOGY   OF   SEWAGE  343 

The  sewage  is  allowed  to  flow  slowly  through  the  tank  and  after  some 
time,  from  a  few  days  to  a  month  or  more,  a  normal  and  constant 
flora  will  have  become  resident  there.  This  flora  will  soon  have  be- 
come so  well  established  that  the  incoming  sewage  laden  with  a  flora 
of  its  own  mingles  with  a  liquid  in  which  the  established  flora  is  so 
greatly  in  excess  that  the  former  in  large  measure  gives  way  to  the 
latter.  In  this  way,  while  the  sewage  itself  moves  onward  and  is 
gone  within  a  few  hours,  the  flora  is  constant  and  persistent.  A  further 
aid  in  preserving  this  constant  flora  is  the  sludge  at  the  bottom,  in 
which  the  bacteria  lodge  and  multiply  and  from  which  they  are  carried 
upward  by  the  ever  moving  eddies  and  constantly  re-inoculate  the 
liquid  above  (Fig.  130). 

THE  DESTRUCTION  or  SEWAGE  BACTERIA 

BY"  BIOLOGICAL  PROCESSES. — Reference  has  already  been  made 
to  the  effect  of  biological  processes  of  purification  upon  pathogenic 
bacteria.  What  was  stated  in  regard  to  the  pathogens  is  equally  true 
of  the  sewage  bacteria  as  a  whole.  Their  destruction  is  due  to  time  and 
an  environment  unfavorable  to  growth,  rather  than  to  any  specific 
cause.  Further  evidence  of  these  facts  may  now  be  given.  Bacteria 
as  a  whole  do  pass  even  the  fine-grained  filters  in  large  numbers. 
Careful  analyses  of  their  types  show  them  to  be  a  haphazard  mixture 
from  the  original  sewage  flora  with  little  or  no  observable  selection. 
Houston  pointed  out  the  relative  abundance  of  the  streptococci,  sup- 
posedly delicate  organisms,  and  found  on  the  whole  that  the  relative 
abundance  of  the  different  kinds  of  bacteria  seemed  to  be  much  the 
same  in  the  effluent  as  in  the  crude  sewage. 

On  the  whole  we  may  conclude  that  the  biological  processes  remove 
bacteria  not  by  any  specific  antagonistic  action  but  by  delaying  their 
passage  and  permitting  the  natural  decrease  that  occurs  when  multi- 
plication is  prevented.  The  more  efficient  the  mechanism  of  the 
filter  in  producing  this  delay  the  more  complete  will  be  the  removal. 

BY  CHEMICAL  PROCESSES. — A  much  more  reliable  and  economical 
method  for  bacterial  destruction  is  now  available  in  chemical  disin- 
fection of  sewage  effluents.  The  writer's  studies  at  Boston,  Baltimore 
and  elsewhere  have  shown  that  the  application  of  hypochlorite  of 
calcium  in  amounts  depending  upon  the  character  of  the  effluent,  and 


344  MICROBIOLOGY   OF   WATER  AND    SEWAGE 

ranging  from  one  to  five  parts  per  million  of  available  chlorine  (25  to 
125  pounds  of  bleaching  powder  per  million  gallons),  will  produce  a 
bacterial  removal  amounting  to  98  or  99  per  cent.  This  disinfectant 
is  the  most  efficient  of  the  known  germicides,  cost  being  considered. 
By  this  means  it  is  possible  to  practically  eliminate  the  bacteria,  good 
and  bad,  from  an  effluent  and  it  is  no  longer  necessary  nor  desirable 
to  seek  high  bacterial  removals  in  the  purification  process  proper. 
By  thus  dividing  the  work  of  purification  into  its  component  parts 
each  part  can  be  carried  out  at  a  maximum  of  efficiency  and  economy. 


DIVISION  III* 

MICROBIOLOGY  OF  SOIL 


CHAPTER  I 
MICROORGANISMS  AS  A  FACTOR  IN  SOIL  FERTILITY 

INTRODUCTION 

Rational  views  on  soil  fertility  were  first  presented,  in  a  systematic 
way,  by  Justus  von  Liebig  in  1840.  In  his  "  Organic  Chemistry  in  its 
Applications  to  Agriculture  and  Physiology"  he  developed  important 
theories  on  the  circulation  of  carbon  and  nitrogen  in  nature,  and  on 
the  function  of  the  so-called  mineral  constituents  of  plants. 

When  Liebig's  book  appeared,  many  of  the  leaders  and  students  of 
agriculture  still  believed  that  humus,  the  partly  decomposed  residues  of 
plants  and  animals  in  the  soil,  was  the  direct  food  of  crops.  They 
believed  that  soils  could  yield  poor  or  rich  harvests  in  proportion  to  the 
amount  of  humus  present  in  them;  they  believed,  in  other  words,  that 
plants,  like  animals,  used  organic  substances  as  food. 

Liebig  rendered  a  great  service  to  agriculture  in  emphasizing  the 
significance  of  decay  processes.  He  made  it  evident  that  humus  as 
such  is  of  no  use  to  plants,  and  that  it  becomes  valuable  only  in  so  far 
as  it  is  resolved  into  the  simple  compounds  carbon  dioxide,  ammonia, 
nitric  acid  and  various  mineral  salts.  To  be  sure,  he  regarded  the 
decomposition  of  organic  matter  as  a  phenomenon  purely  chemical, 
nevertheless  he  succeeded  in  showing  that  decay,  putrefaction  and 
fermentation  are  fundamental  facts,  connecting  links  between  the 
world  of  the  living  and  the  world  of  the  dead. 

The  research  of  the  following  decades  brought  to  light  the  intimate 
relation  existing  between  microorganisms  and  the  decomposition  of 

*  Prepared  by  Jacob  G.  Lipman  with  exception  of  sub-chapter  on  "Soil  Inoculation"  which 
has  been  prepared  by  S.  F.  Edwards.  The  author  is  indebted  to  Dr.  S.  A.  Waksman  for  help 
in  the  revision  of  a  portion  of  the  manuscript. 

345 


346  MICROBIOLOGY   OF   SOIL 

organic  matter.  In  the  realm  of  soil  fertility  the  new  discoveries  re- 
vealed the  vastness  of  the  task  assigned  to  soil  microorganisms  in 
providing  available  food  for  crops.  It  was  shown  that  under  the  attack 
of  bacteria  and  of  other  microorganisms  the  various  organic  debris  in 
the  soil  is  split  into  relatively  small  chemical  fragments;  that  the 
carbon  is  restored  to  the  air  as  carbon  dioxide;  that  the  nitrogen  is 
changed  into  ammonia,  nitrites  and  nitrates.  It  was  shown,  further, 
that  in  this  breaking  down  of  organic  matter  the  various  cleavage 
products,  and,  particularly,  carbon  dioxide,  hasten,  to  an  amazing 
extent,  the  weathering  of  the  rock  particles  and  make  available  thereby 
the  mineral  portion  of  plant  food.  It  was  shown,  likewise,  that  apart 
from  accomplishing  the  transformation  of  unavailable  into  available 
plant  food,  microorganisms  are  concerned  also  in  the  addition  of 
nitrogen  compounds  to  the  soil.  The  evidence  gathered  slowly  by 
many  investigators  made  it  plain,  therefore,  that  microbes  are  an 
important  factor  in  the  growing  of  cultivated  and  uncultivated  plants. 
Hence,  the  important  place  assigned  to  microorganisms  in  the  study  of 
soil  fertility  problems. 

THE  SOIL  AS  A  CULTURE  MEDIUM 

Arable  soils  present  so  wide  a  range  of  conditions  as  to  modify, 
materially,  the  development  and  predominance  of  different  species. 
Variations  as  to  moisture,  temperature,  aeration,  reaction,  food  supply 
and  biological  relations  are  important,  in  each  case,  in  determining 
the  survival  or  disappearance  of  any  particular  species.  For  this 
reason,  the  study  of  soil  microorganisms  must  reckon  with  the  mechan- 
ical composition  of  soils,  their  ability  to  retain  water  and  their  content 
of  inert  and  soluble  plant  food. 

MOISTURE  RELATIONS  IN  THE  SOIL 

AMOUNT  AND  DISTRIBUTION  OF  RAINFALL. — Precipitation  in 
different  regions  of  the  earth's  surface  varies  from  practically  nothing 
to  more  than  1,524  cm.  (600  inches)  per  annum.  A  portion  of  this 
water  runs  off  the  surface  into  the  nearest  stream,  another  portion  is 
rapidly  changed  into  vapor  and  is  returned  to  the  atmosphere,  and  the 
remainder  passes  downward,  into  the  soil  and  becomes  the  medium 
in  which  plant  food  is  dissolved.  It  is  estimated  that  only  about  half 


MICROORGANISMS   AS   A   FACTOR   IN   SOIL  FERTILITY          347 

the  total  rainfall  percolates  through  the  soil.  Where  the  soils  are 
open  and  nearly  level  the  proportion  of  percolating  water  is  relatively 
greater;  where  the  soils  are  fine-grained  and  more  or  less  impervious, 
or  the  topography  broken,  the  proportion  is  relatively  smaller. 

Bacteria  and  other  microorganisms,  as  well  as  the  higher  plants,  are 
directly  influenced  by  the  amount  of  moisture  available  for  their  various 
needs.  Hence  soil  microbial  activities  are  affected  not  alone  by  the 
amount  of  rainfall,  but  also  by  its  distribution.  It  is  obvious,  for 
instance,  that  an  annual  rainfall  of  762  mm.  (30  inches)  distributed 
rather  uniformly  throughout  the  year  would  produce  different  soil- 
moisture  relations  than  the  same  amount  of  precipitation  confined  to 
only  two  or  three  months.  As  is  pointed  out  by  Abbe,  a  daily  pre- 
cipitation of  2  mm.  (.079  inch)  distributed  throughout  the  three 
summer  months  would  be  quickly  changed  into  vapor,  and  would 
hardly  wet  the  soil;  whereas  the  total  quantity  of  180  mm.  (7  inches) 
evenly  divided  into  ten  or  twelve  rains  would  penetrate  the  soil  to  a 
considerable  depth,  and  would  furnish  very  favorable  conditions  for 
microbial  development.  In  a  similar  manner  it  is  pointed  out  by  Hil- 
gard  that  Central  Montana,  and  the  region  in  the  vicinity  of  the  bay 
of  San  Francisco,  have  each  a  total  precipitation  of  610  mm.  (24  inches). 
But  while  in  Montana  the  rainfall  is  distributed  over  the  entire  year 
and  irrigation  becomes  necessary,  the  precipitation  near  San  Francisco 
is  limited  to  the  portion  of  the  year  that  nearly  coincides  with  the 
growing  season,  and  crops  are  enabled  to  mature  without  irrigation. 

RANGE  or  SOIL  MOISTURE. — Any  given  volume  of  dry  soil  consists 
of  solid  particles  separated  by  empty  spaces.  The  sum  of  these  spaces 
is  known  as  the  "  pore-space."  It  varies  from  about  one-third  of  the 
entire  volume  in  coarse  sands  to  more  than  two-thirds  in  pipe  clay.  In 
peat  and  muck  it  may  amount  to  as  much  as  80  or  90  per  cent  of  the 
entire  volume.  Under  air-dry  conditions  each  soil  grain  is  surrounded 
by  a  very  thin  film  of  moisture  designated  as  hygroscopic  water.  When 
air-dry  soil  is  moistened  the  films  around  the  soil  particles  become 
thicker  and  finally  cease  to  be  isolated.  A  continuous  liquid  membrane, 
as  it  were,  is  stretched  from  particle  to  particle,  and  the  surface  tension 
that  thus  comes  into  play  is  capable  of  lifting  large  amounts  of  water 
to  the  surface.  The  continuous  film  of  soil  water  that  can  hold  its 
own  against  the  pull  of  gravity  is  known  as  capillary  water.  Finally, 
when  the  liquid  films  around  the  soil  grains  increase  in  thickness  be- 


348  MICROBIOLOGY  OF   SOIL 

yond  a  certain  point,  the  attraction  between  the  molecules  in  the  soil 
grains  and  the  more  distant  molecules  of  water  is  no  longer  great 
enough  to  overcome  the  force  of  gravitation,  and  the  excess  of  water 
percolates  downward.  The  water  more  or  less  readily  moved  by 
gravitation  is  called  hydrostatic  water. 

For  any  given  conditions  of  the  soils  the  amount  of  hydrostatic, 
capillary  and  hygroscopic  water  is  directly  dependent  on  the  mechanical 
structure.  It  is  evident  that  the  aggregate  surface  of  the  particles  in 
a  fine-grained  soil  is  much  greater  than  that  in  a  coarse-grained  soil. 
Actual  determinations  have  shown  that  the  aggregate  inner  surface 
of  .02832  c.m.  (r  cu.  ft.)  of  coarse  sand  may  be  but  a  fraction  of  an 
acre;  whereas  the  same  quantity  of  the  finest  clay  may  have  an 
inner  surface  equivalent  to  1.2141-1.6188  hectares  (3  or  4  acres). 
These  differences  are  to  be  expected,  since,  as  is  shown  by  Lyon  and 
Fippin,  i  g.  of  fine  gravel  may  contain  252  particles;  i  g.  of  medium 
sand,  13,500  particles;  i  g.  of  very  fine  sand,  1,687,000  particles;  i  g. 
of  silt,  65,100,000  particles,  and  i  g.  of  clay,  45,500,000,000  particles. 

Since  the  soil  water  is  spread  as  a  film  over  the  solid  particles  and 
varies  in  amount  with  the  fineness  or  coarseness  of  the  soil,  and  since 
the  quantity  of  plant  food  going  into  solution  is  determined  largely 
by  the  amount  of  water  in  contact  with  the  soil  particles,  it  follows  that 
clay  soils  will,  under  the  same  conditions,  contain  more  plant  food  in 
solution  than  loam  soils  and  still  more  than  sandy  soils.  From  the 
standpoint  of  soil  microbiology  this  is  important,  for  the  microorganisms 
live  and  multiply  in  the  film  water  surrounding  the  soil  particles.  The 
concentration  of  salts  in  this  film  water  as  well  as  their  composition 
must  of  necessity  affect  bacterial  activities.  In  the  same  way,  methods 
of  tillage  and  cropping  affecting  the  concentration  and  composition 
of  the  film  water  will  modify  the  chemical  changes  caused  by  bacteria 
and  other  microorganisms. 

EFFECT  OF  DROUGHT  AND  OF  EXCESSIVE  MOISTURE. — Optimum 
conditions  for  plant  growth  and  the  development  of  many  important 
soil  bacteria  are  furnished  when  about  half  of  the  entire  pore  space  is 
filled  with  water.  In  light  sandy  soils  the  optimum  moisture  content 
may  be  reached  when  the  wet  material  contains  scarcely  more  than  8 
to  10  per  cent  of  water  by  weight;  while  in  silt  and  clay  soils  the 
optimum  may  reach  1 6  to  20  per  cent  or  even  more. 

Continued  depletion  of  soil  moisture  by  plant  roots  and  evaporation 


MICROORGANISMS   AS   A   FACTOR   IN   SOIL  FERTILITY          349 

at  the  surface  causes  the  film  of  capillary  water  to  stretch  more  and 
more.  Finally  it  becomes  very  thin,  breaks,  and  ceases  to  be  con- 
tinuous. The  soil  then  becomes  air-dry  and  contains  only  hygro- 
scopic water.  It  is  estimated  by  Lyon  and  Fippin  that,  under  average 
conditions  of  humidity,  light  sand  will  contain  0.5  to  i  per  cent  of 
hygroscopic  moisture;  silt  loam,  2  to  4  per  cent;  and  clay,  8  to  12  per  • 
cent.  The  amount  of  water  present  in  air-dry  muck  or  peat  may  range 
up  to  40  per  cent,  or  even  more.  According  to  Hall  the  film  of  hygro- 
scopic moisture  is  about  0.75^  (0.00003  inch)  thick.  As  the  soil 
dries  out  bacterial  activity  is  suspended  and  many  vegetative  cells 
undoubtedly  perish.  Nevertheless,  it  will  be  seen  that  the  moisture 
film  even  in  air-dry  material  is  deep  enough  to  allow  the  bacteria  a 
reasonable  degree  of  protection.  This  will  account  for  the  survival 
of  non-spore-bearing  bacteria  in  dry  soil  for  a  long  time.  Indeed,  in- 
stances are  on  record  of  the  isolation  of  Azotobacter  and  Nitrosomonas 
from  soils  that  had  been  kept  in  a  dry  state  in  the  laboratory  for 
several  years.  It  may  be  noted,  in  this  connection,  that  in  the  process 
of  drying  the  soluble  salts  in  the  soil  the  moisture  may  be  sufficiently 
concentrated  in  the  films  to  cause  plasmolysis  and  the  destruction 
of  individual  cells. 

On  the  other  hand,  excessive  moisture  in  the  soil  is  not  only  directly 
unfavorable  to  aerobic  species  in  that  it  limits  their  supply  of  oxygen, 
but  is  objectionable  because  it  encourages  the  formation  of  reduction 
products  that  are  toxic  to  these  species.  It  is  apparent,  therefore,  that 
favorable  conditions  for  the  formation  of  available  plant  food  by 
bacteria  are  created  when  a  certain  relation  is  established  between  the 
volumes  of  moisture  and  air  in  the  soil.  The  shifting  of  this  relation  in 
one  direction  or  another  is  bound  to  react  on  species  relationships  and 
numbers. 

COLLOIDAL  NATURE  OF  THE  SOIL.- — The  colloidal  condition  of 
the  soil  imparts  to  it  the  ability  to  absorb  substances  from  their 
solutions  as  well  as  the  ability  to  change  them  from  a  flocculated 
to  a  deflocculated  state.  Another  important  colloidal  property  of 
soil  is  the  formation  of  a  colloidal  solution  in  pure  water  and  coagu- 
lation by  the  addition  of  small  quantities  of  electrolytes.  Soluble 
fertilizers  when  added  to  the  soil  are  adsorbed  by  the  latter:  other- 
wise, they  could  easily  be  washed  out  by  drainage.  The  adsorbed 
substances  displace  others  which  may  be  washed  out  of  the  soil. 


35°  MICROBIOLOGY  OF   SOIL 

The  addition  of  ammonium  and  potassium  salts,  for  example,  re- 
sults in  the  displacement  of  the  corresponding  calcium  salts,  which  can 
be  washed  out,  and  the  formation  of  insoluble  nitrogen  or  potassium 
compounds  which  remain  in  the  soil.  On  adding  sodium  and  magnes- 
ium salts  to  the  soil,  displacement  of  some  of  the  insoluble  potassium 
salts  may  take  place  and  these  may  become  available  for  plant  growth. 
The  interchanges  taking  place  between  the  salts  existing  in  the  soil 
and  those  added  in  the  form  of  fertilizers  have  an  important  effect  upon 
soil  biological  phenomena  and  plant  nutrition.  On  heating  or  drying 
soils,  an  increase  in  the  amount  of  soluble  food  is  produced  which  is 
probably  a  result  of  the  change  produced  in  the  colloids.  It  is  in  this 
colloidal  complex  of  organic  and  inorganic  compounds,  saturated  with 
water  and  surrounded  by  the  mineral  particles  that  most  of  the  soil 
biological  phenomena  take  place. 

AERATION 

MECHANICAL  COMPOSITION  OF  SOILS. — Soil  ventilation  is  an  impor- 
tant factor  in  crop  production.  It  provides  for  the  proper  supply  of 
elementary  oxygen  so  essential  to  decomposition  processes  in  normal 
soils;  for  the  supply  of  elementary  nitrogen  required  by  nitrogen-fixing 
species;  for  the  removal  of  excessive  amounts  of  carbon  dioxide;  and 
for  the  destruction  of  various  toxic  substances.  The  intimate  relation 
existing  between  soil  ventilation  and  the  mechanical  composition  of  the 
soil  material  is  bound  to  react  on  the  microbial  factors  involved.  It  is 
well  known  that  the  rate  of  flow  of  air  through  soils  is  inversely  propor- 
tional to  the  fineness  of  the  material;  in  other  words,  the  fine-grained 
soils,  notwithstanding  their  greater  pore  space,  will  not  allow  air  to 
pass  through  them  as  rapidly  as  coarse-grained  soils.  King  shows,  for 
instance,  that  5,000  c.c.  of  air  passed  through  a  column  of  fine  gravel 
in  thirty-seven  seconds,  whereas  in  similar  columns  of  medium  sand, 
fine  sand,  loam  and  fine  clay  soil  the  same  amount  of  air  required  for  its 
passage  1,178,  44,310,  282,200,  and  2,057,000  seconds  respectively. 

AEROBIC  AND  ANAEROBIC  ACTIVITIES. — The  more  rapid  diffusion 
of  gases  from  open  soils  naturally  leads  to  a  more  frequent  renewal  of 
their  oxygen  supply.  In  its  turn,  the  latter  affects  the  ratio  of  aerobes 
to  anaerobes;  it  follows,  therefore,  that  in  clay  soils  and  clay  loam  soils 
the  activities  of  aerobic  species  are  retarded  to  a  greater  extent  than 
they  are  in  sandy  loams  or  sandy  soils.  It  follows,  also,  that  in  fine- 


MICROORGANISMS   AS   A   FACTOR   IN   SOIL   FERTILITY          351 

grained  soils  the  activities  of  the  aerobes  are  confined  to  a  shallower 
soil  layer  than  in  coarser  grained  soils.  The  reverse  is  true  of  anaerobic 
species.  Methods  of  soil  treatment  tending  to  improve  soil  ventilation 
react  both  on  the  amount  of  chemical  change  produced  by  definite 
species,  as  well  as  the  numerical  ratio  of  different  species  to  one  another. 
Among  such  methods  may  be  included  drainage,  liming,  manuring  and 
tillage. 

RATE  OF  OXIDATION  OF  CARBON,  HYDROGEN  AND  NITROGEN. — 
Experiments  carried  out  by  Wollny  proved  conclusively  that  the  pro- 
duction of  carbon  dioxide  in  soils  is  directly  affected  by  the  amount  of 
oxygen  supplied;  that  is,  by  the  more  or  less  thorough  aeration  of  the 
soil.  In  one  of  these  experiments  air  containing  varying  proportions 
of  oxygen  and  nitrogen  was  passed  through  columns  of  soil.  When 
this  air  contained  21  per  cent  of  oxygen  there  were  produced  for  every 
i,ooo  volumes  of  air  12.51  volumes  of  carbon  dioxide;  while  with  2  per 
cent  of  oxygen  in  the  entering  air  there  were  produced  only  3.62 
volumes  of  carbon  dioxide.  Similar  observations  were  made  by 
Schloesing  in  connection  with  the  formation  of  carbon  dioxide  and  of 
nitric  acid.  Deherain  and  many  others  have  recorded  the  favorable 
influence  of  aeration  on  the  rate  of  nitrate  formation,  while  Lipman 
and  Koch  have  observed  an  increased  fixation  of  nitrogen  by  Azotobacter, 
consequent  upon  a  better  supply  of  oxygen. 

THE  MINERALIZATION  OF  ORGANIC  MATTER. — Conditions  that  favor 
the  intense  activities  of  decay  bacteria  lead  to  a  relatively  rapid  restora- 
tion of  the  phosphorus,  sulphur,  calcium,  magnesium  and  potassium 
that  had  been  made  fast  in  plant  tissues,  to  the  stock  of  available  plant 
food  in  the  soil;  indeed,  in  extremely  well-aerated  soils  the  decomposition 
of  organic  matter  and  its  ultimate  mineralization  proceed  too  fast.  It 
often  happens  that  the  farmer  is  unable  to  maintain  a  proper  supply 
of  humus  in  these  soils  because  of  their  openness  and  is  forced  to  adopt 
measures  that  will  retard  soil  aeration.  He  resorts  therefore,  to  rolling, 
marling,  manuring  and  green  manuring. 

On  the  other  hand,  heavy,  fine-grained  soils  are  not  sufficiently  well 
aerated  to  allow  a  rapid  mineralization  of  the  organic  matter.  Under 
extreme  conditions  the  decomposition  processes  do  not  keep  pace  with 
the  process  making  toward  the  accumulation  of  organic  matter,  and  a 
more  or  less  considerable  increase  in  the  amount  of  the  latter  takes 
place.  This  occurs  in  low  lying  meadows,  and,  more  particularly,  in 


352 


MICROBIOLOGY    OF    SOIL 


bogs  and  swamps.  Hence  the  farmer  attempts  to  intensify  aeration 
and  the  resulting  mineralization  of  the  humus  by  more  thorough 
tillage,  drainage,  liming  and  manuring. 

TEMPERATURE 

INFLUENCE  OF  CLIMATE  AND  SEASON. — An  illustration  of  the  differ- 
ences that  may  exist  in  the  soil  temperatures  of  different  regions  is  given 
by  a  comparison  of  the  mean  temperatures  of  1901  recorded  at  Moscow, 
Idaho,  and  New  Brunswick,  New  Jersey.  The  soil  temperatures  were 
taken  to  a  depth  of  152  mm.  (6  inches). 

SOIL  TEMPERATURE,*  1901 


Jan. 

Feb. 

Mch. 

Apr. 

May 

June 

July 

Aug. 

Sept. 

Oct. 

Nov. 

Dec. 

I 

Moscow,  Idaho.  .  . 

32.0 

30.0 

35.0 

40.0 

52.0 

58.0 

68.0 

72.0 

57.0 

50.0 

40.0 

34-0 

New      Brunswick, 

3i.S 

28.6 

35.3 

47.9 

57.9 

72.1 

76.4 

73-4 

68.5 

56.0 

41.1 

33-4 

N.J. 

AIR  TEMPERATURE,*  1901 


Moscow,  Idaho.  .  . 

30.0 

30.5 

38.3 

44-0 

56.9 

55-0 

65.5 

69.6 

50.3 

50.5 

39-5 

39-0 

New    Brunswick, 

30.8 

24.8 

39.1 

48.3 

59-2 

70.9 

77-4 

74-6 

67.6 

54-6 

38.6 

32.6 

N.J. 

It  will  be  observed  that  in  the  months  of  November  to  March  the 
soil  temperatures  in  the  two  places  were  nearly  the  same.  On  the 
other  hand,  in  April  to  October  the  average  temperatures  at  New 
Brunswick  were  for  soil  14.5°  (s8°F.)  and  for  air  22.5°  (72°?.), re- 
spectively; and  in  July  they  were  20.0°  (68°F.)  and  24.5°  (y6.4°F.) 
respectively.  It  will  also  be  observed  that  there  is  an  unmistakable 
relation  between  the  corresponding  air  and  soil  temperatures. 

As  a  further  illustration  of  the  relation  of  climate  to  temperature  a 
comparison  may  be  made  of  the  average  daily  mean  temperatures  at 
Bismarck,  North  Dakota,  for  the  period  1873-1895,  and  at  Key  West, 
Florida,  for  the  period  1872-1895. 

DAILY  MEAN  TEMPERATURES*  (Am) 


Jan. 

Feb. 

Mch. 

Apr. 

May    June 

July 

Aug. 

Sept. 

Oct. 

Nov. 

Dec. 

Bismarck,  N.  D... 

4-5 

9-5 

22.6 

42.1 

54-2 

63.8 

69.5 

67.5 

57-0 

43.8 

25.9 

14.7 

Key  West,  Fla  

69-7 

71.4 

72.7 

76.1 

79-4 

82.5 

83-9 

83.9 

82.  5 

78.5 

74-2 

70.0 

*  Recorded  in  Fahrenheit  scale. 


MICROORGANISMS   AS   A   FACTOR   IN   SOIL   FERTILITY 


353 


It  is  obvious  from  the  figures  given  here  that,  because  of  the  im- 
portant temperature  variations  of  different  soil  regions,  the  micro- 
biological activities  must  be  profoundly  modified.  But  apart  from  the 
climatic  variations  already  indicated  there  are  seasonal  variations  in 
any  particular  locality  that  are  of  great  moment  for  soil  microbiological 
activities.  Such  differences  are  demonstrated  by  the  temperatures 
of  1898  and  1902,  taken  to  a  depth  of  152  mm.  (6  inches),  at  New 
Brunswick,  N.  J. 

SOIL  TEMPERATURES* 


Jan. 

Feb. 

Mch. 

Apr. 

May 

June 

July 

Aug. 

Sept. 

Oct. 

Nov. 

Dec. 

New  Brunswick, 

N.  J.  (1898)  ... 

33.2 

33.1 

45-  1 

48.9 

59.1 

76.0 

79-3 

77.8 

72.0 

60.  1 

44-6 

33.6 

New  Brunswick, 

N.  J.  (1902).... 

30.7 

28.9 

41-3 

49.5 

60.4 

68.0 

72.6 

70.5 

65.9 

56.4 

48.6 

34-1 

In  this  instance,  the  season  of  1898  was  not  only  earlier,  but  the 
temperatures  of  June  to  September  were  sufficiently  higher  to  favor 
more  intense  bacterial  growth  and  activity. 

EARLY  AND  LATE  SOILS. — Under  any  given  climatic  conditions  the 
warming  up  of  soils  in  the  spring  will  depend  on  their  chemical  and 
mechanical  composition,  color,  tillage  and  topography.  Because  of  the 
high  specific  heat  of  water,  fine-grained  soils  containing  a  relatively 
large  amount  of  moisture  will  warm  up  more  slowly  than  coarse-grained 
soils  containing  a  relatively  small  amount  of  moisture.  The  differences 
in  the  specific  heat  of  humus,  sand,  clay  and  chalk  are  less  important, 
yet  they  introduce  appreciable  variations  in  the  soil  temperature 
according  to  the  proportion  of  each  present.  The  topography  of  the 
soil  introduces  a  factor  of  some  importance  for  it  affects  the  inclina- 
tion toward  the  sun's  rays  as  well  as  the  drainage  conditions.  Tillage 
operations  are  of  considerable  moment,  since  they  influence  the  rate 
of  evaporation,  that  is,  the  rate  at  which  heat  is  lost  from  the  soil  by 
the  transformation  of  liquid  water  into  vapor.  Finally  the  color  of 
soils  exerts  an  influence  on  their  temperature  in  that  it  affects  the 
absorption  and  reflection  of  heat. 

Taking  all  of  the  factors  together,  it  is  found  that  sandy  soils  and 
sandy  loams  are  early  soils,  because  they  part  readily  with  their  excess 

*  Recorded  in  Fahrenheit  scale. 
23 


354  MICROBIOLOGY   OF   SOIL 

of  water.  Clay  soils  and  clay  loams  are,  on  the  other  hand,  late  soils; 
it  means,  therefore,  that  in  the  more  open  soils  microbial  activities  be- 
come intense  earlier  in  the  spring.  Market  gardeners  usually  attempt 
to  improve  matters  still  further  by  the  use  of  large  quantities  of  readily 
fermentable  manure  that  develops  enough  heat  to  raise  slightly  the 
soil  temperature. 

PRODUCTION  AND  ASSIMILATION  or  PLANT  FOOD. — It  was  observed 
by  Moller  that  slight  amounts  of  carbon  dioxide  may  be  evolved 
from  frozen  soil.  Kostychev  could  detect  a  considerable  production 
of  carbon  dioxide  at  o°  to  5°.  In  a  series  of  experiments  carried  out 
by  Wollny  the  amounts  of  carbon  dioxide  produced  were  as  follows : 

CO2  IN  1,000  VOLS.  or  AIR 

Water  in  soil  10°  20°  30°  40°  SO0 


6.79  per  cent  

2.03 

3-22 

6.86 

14.69 

25.17 

26  79  per  cent. 

18.38 

<4.  22 

63.  tjo 

80.06 

81.52 

46  79  per  cent 

•zer   07 

6l    40 

82.  12 

91.86 

07  .48 

The  increased  production  of  carbon  dioxide  at  the  higher  temperatures, 
as  shown  in  the  above  table,  corresponded  with  the  observations  that  had 
already  been  made  by  Ebermayer,  Schloesing  and  others,  that  carbon 
dioxide  production  in  the  soil  is  greater  in  summer  than  it  is  in  winter. 
These  facts,  taken  together  with  the  early  observations  of  Forster  on 
the  multiplication  of  photo-bacteria  at  o°,  and  the  more  recent  ob- 
servations of  numerous  investigators  on  the  multiplication  of  in- 
dividual species,  or  of  mixtures  of  species  in  milk,  water,  soil,  butter, 
etc.,  at  o°,  or  even  below  that,  make  it  evident  that  bacterial  activities 
are  not  entirely  suspended  at  relatively  low  temperatures.  As  the 
latter  rises  these  activities  become  more  intense  as  gauged  by  the 
formation  of  carbon  dioxide. 

Coming  down  to  specific  groups  of  soil  bacteria,  it  may  be  noted  that 
at  12°  nitrification  is  already  quite  perceptible;  that  urea  bacteria  grow 
slowly  at  5°;  Ps.  radicicola  at  4°;  members  of  the  B.  subtilis  group  at 
6°  to  10°,  etc.  At  15°  the  breaking  down  of  organic  matter  is  fairly 
rapid,  and  at  25°  the  optimum  is  reached  for  many  species.  It  follows, 
thus,  that  the  production  of  plant  food — namely,  ammonia,  nitrates, 
sulphates,  phosphates,  etc. — gains  rapid  headway  as  the  optimum  tem- 
peratures are  approached.  The  organic  matter  itself,  apart  from  serv- 


MICROORGANISMS   AS   A   FACTOR    IN   SOIL   FERTILITY          355 

ing  as  a  source  of  plant  food,  furnishes  carbon  dioxide  and  various 
organic  acids  that  help  to  attack  the  rock  fragments  and  to  render 
available  compounds  of  phosphorus,  potassium,  calcium  and  mag- 
nesium. It  is  likewise  evident  that  in  warm  countries  bacterial 
activities  are  not  only  more  intense  at  any  one  time,  but  they  continue 
through  a  longer  period.  For  this  reason,  the  soils  of  the  South  can 
furnish  both  relatively  and  absolutely  a  greater  amount  of  available 
plant  food  than  the  soils  of  the  North. 

The  production  of  plant  food  is  necessarily  followed  by  more 
vigorous  growth  of  bacteria  and  of  higher  plants.  More  food  is,  there- 
fore, assimilated  and  more  moisture  used  up  until  the  very  rank  growth 
of  the  crops  hastens  the  depletion  of  the  soil  moisture.  In  this  manner 
the  soil  may  be  dried  out  sufficiently  to  retard  seriously  the  growth  of 
soil  bacteria  and  to  retard  thereby  the  decompositon  of  organic  matter; 
under  such  conditions,  moisture,  rather  than  temperature,  becomes 
the  controlling  factor  of  growth. 

REACTION 

RANGE  OF  SOIL  ACIDITY. — Acid  soils  are  very  common  in  humid 
regions.  The  older  soils  of  Europe  include  extensive  areas  whose  lime 
content  has  been  restored  repeatedly  by  the  application  of  wood  ashes, 
marl,  oyster  and  clam  shells,  and  various  grades  of  burned  or  crushed 
limestone.  In  the  United  States  acidity  is  becoming  prevalent  in  many 
of  the  cultivated  soils,  as  is  shown  by  the  investigations  of  the  Rhode 
Island,  Ohio,  Illinois,  Oregon  and  Florida  experiment  stations.  These 
investigations,  confirmed  by  experiments  in  other  states,  show  that 
there  is  a  marked  removal  of  lime  and  of  other  basic  materials  from  the 
soil  as  cultivation  and  the  use  of  commercial  fertilizers  become  more 
thorough.  Knisley  shows,  for  instance,  that  38.75  per  cent  of  the 
Oregon  soils  examined  were  acid,  and  that  16.25  per  cent  were  strongly 
acid.  Similarly,  Blair  found  that  of  189  samples  of  different  Florida 
soils  and  subsoils  examined,  68.22  per  cent  of  the  former  and  51.35 
per  cent  of  the  latter  were  acid.  He  also  found  that  virgin  soils  were 
less  acid  than  cultivated  soils. 

CAUSES  OF  SOIL  ACIDITY. — Soil  acidity  may  be  due  to  acids  or  acid 
salts,  both  inorganic  and  organic.  Under  ordinary  conditions  the 
latter  are  of  much  greater  importance  than  the  former  as  a  cause  of 
soil  acidity.  This  is  demonstrated  by  the  extremely  acid  conditions 


MICROBIOLOGY   OF   SOIL 

of  peat  and  muck  soils  that  are  particularly  rich  in  organic  acids.  In 
soils  left  to  themselves  the  formation  of  basic  substances  in  the  break- 
ing down  of  silicates  and  other  compounds  keeps  pace  with  their 
neutralization  by  acid  and  their  removal  in  the  drainage  water.  When 
soils  are  placed  under  cultivation,  lime  and  other  bases  are  removed 
more  rapidly  and  the  inert  humic  acids  are  left  behind.  The  loss  of 
bases  is  intensified  by  application  of  acid  phosphate,  potash  salts  and 
ammonium  sulphate,  commonly  used  as  fertilizers.  This  accounts 
for  the  less  extensive  acidity  in  and  among  virgin  soils  as  compared 
with  cultivated  soils.  Arid  soils  lose  scarcely  any  of  their  basic  sub- 
stances by  leaching  and  are  seldom  acid.  Residual  limestone  soils 
may  be  alkaline,  neutral  or  acid,  according  to  the  loss  of  bases  they 
have  suffered  by  leaching.  Low-lying  soils,  including  meadows 
and  swamps  may  accumulate  large  amounts  of  organic  acids  because 
of  their  imperfect  aeration. 

The  more  recent  investigations  of  the  nature  of  soil  acidity  have 
suggested  a  physical  explanation,  namely,  that  the  acidity  of  the 
soil  is  due  not  to  the  existence  of  definite  humic  and  other  complex 
organic  acids,  but  rather  to  selective  adsorption.  According  to  some 
investigators  there  is  a  direct  adsorption  of  the  base  when  a  soil  is 
treated  with  a  salt  solution.  Hence,  the  behavior  of  the  soil  towards 
neutral  salts  is  not  due  to  the  presence  of  organic  matter,  but  to  inor- 
ganic compounds,  probably  hydrated  silicates.  According  to  others  the 
development  of  acidity  in  the  salt  solution  is  due  rather  to  an  exchange 
of  bases :  aluminum  is  given  up  from  the  soil  in  amounts  approximately 
equivalent  to  the  base  adsorbed. 

Through  the  action  of  microorganisms  in  the  soil,  the  organic  matter 
is  decomposed  with  the  liberation  of  weak  organic  acids  (oxalic,  citric, 
CO2,  etc.).  '  By  the  interaction  of  these  acids  in  the  soil  solution  and 
the  basic  material  held  adsorbed  by  the  soil,  soluble  salts  are  formed 
which  are  subsequently  removed  by  leaching:  the  soil  can  then  adsorb 
more  basic  material,  giving  rise  to  soil  acidity. 

SOIL  REACTION  AND  HYDROGEN-ION  CONCENTRATION. — The  dif- 
ferent methods  for  measuring  the  lime  requirements  of  soils  are  merely 
attempts  to  measure  the  total  soil  acidity,  but  not  the  intensity  of  the 
acidity  or  the  active  acidity.  The  latter  can  only  be  determined  by 
measuring  the  hydrogen-ion  concentration  of  the  soil.  Pure  water 
dissociates,  producing  equal  concentrations  of  H  ions  and  OH  ions 


MICROORGANISMS    AS    A    FACTOR   IN    SOIL   FERTILITY          357 

indicating  neutrality.  The  product  of  the  concentrations  of  these  ions 
(water)  is  constant,  about  i  X  io~14.  If  the  concentration  of  H  ions  is 
greater  than  that  of  the  OH  ions,  the  solution  is  then  acid.  When  the 
concentration  of  OH  ions  is  greater  than  of  H  ions,  the  solution  is  alkaline. 
Total  acidity  (or  potential  acidity,  to  use  the  expression  of  Sharp  and 
Hoagland)  or  alkalinity  may  be  due  to  undissolved  substances  or  to 
soluble  compounds  only  partly  hydrolyzed  or  dissociated. 

The  hydrogen-ion  concentration  of  the  soil  can  be  measured  both 
electrometrically  ancf  colorimetrically.  The  work  of  Gillespie,  Sharp 
and  Hoagland  and  others  has  brought  out  the  fact  that  soils  vary  greatly 
in  the  hydrogen-ion  concentration,  from  a  high  acidity  to  a  high  alkalin- 
ity. There  is  a  definite  correlation  between  the  hydrogen-ion  concen- 
tration of  soils  and  the  occurrence  and  activities  of  microorganisms. 
Gillespie  has  shown  that  potato  scab  (Actinomyces  scabies)  rarely  occurs 
in  soils  having  a  hydrogen-ion  exponent  lower  than  4.8  to  5.2. 

Gainey  called  attention  to  the  fact  that  Azotobacter  occurs  in  soils 
having  a  hydrogen-ion  exponent  greater  than  6.0,  while  the  more  acid 
soils  are  practically  free  from  this  important  group  of  nitrogen-fixing 
organisms.  Waksman  demonstrated  the  occurrence  of  Azotobacter 
in  cranberry  soils  that  received  an  application  of  lime  and  gave  a  de- 
cided increase  in  crop,  while  the  unlimed  soil  was  too  acid  for  the  organ- 
isms to  act  in;  this  limiting  reaction  for  Azotobacter  corresponded  to  a 
hydrogen-ion  exponent  of  about  6.0. 

CHANGE  OF  REACTION  PRODUCED  BY  MICROORGANISMS  IN  THE 
MEDIUM.- — Microorganisms  modify  the  reaction  of  the  medium  both 
by  their  ability  to  produce  organic  and  inorganic  acids  (in  the  case 
of  sulphur  oxidizing  bacteria)  and  also  by  their  utilization  of  the 
organic  acids  as  sources  of  energy. 

EFFECT  OF  REACTION  ON  NUMBERS  AND  SPECIES. — Some  of  the 
important  groups  of  soil  bacteria  including  nitro,  azoto  and  ammonify- 
ing species  will  develop  slowly  or  not  at  all,  when  the  amount  of  acid  in 
the  medium  is  increased  beyond  a  certain  point.  Hence  it  is  realized 
by  progressive  farmers  that  a  proper  supply  of  lime  is  essential  for  the 
satisfactory  decomposition  of  organic  matter  in  the  soil,  and  the  abund- 
ant supply  of  available  nitrogen  compounds,  as  well  as  of  other  con- 
stituents of  plant  food  to  growing  crops.  The  influence  of  lime  on  the 
multiplication  of  soil  bacteria  is  well  illustrated,  for  instance,  by  the 
experiments  of  Fabricius  and  von  Feilitzen.  These  investigators  found 


MICROBIOLOGY   OF   SOIL 

only  138,500  bacteria  per  g.  in  newly  broken  and  unlimed  peat  soils; 
whereas  in  similar  soils  that  had  been  limed  and  cultivated  for  several 
years  the  numbers  averaged  about  7,000,000  per  g.  and  reached  a 
maximum  of  22,132,000  per  g. 

FOOD  SUPPLY 

ORGANIC  MATTER. — It  may  be  said  truly  that  a  soil  devoid  of 
organic  matter  is  practically  devoid  of  bacteria.  To  the  fresh  and  the 
partially  decomposed  organic  matter  (humus)  the  soil  organisms  must 
look  for  most  of  their  food  and  energy.  Being  largely  of  plant  origin 
this  organic  matter  contains  starches,  fats,  organic  acids,  higher  al- 
cohols, proteins  and  amino-compounds.  Because  of  the  different 
relations  that  these  vegetable  substances  bear  to  the  several  species  of 
soil  bacteria,  a  high  or  low  proportion  of  starch,  of  cellulose,  or  protein 
must  necessarily  modify  both  numbers  and  species  relationships.  For 
instance,  observations  have  been  made  by  Coleman  and  others  that 
small  amounts  of  dextrose  favor  nitrification,  whereas  larger  quantities 
retard  it;  similarly,  it  has  been  noted  that  in  the  spontaneous  de- 
composition of  protein  bodies  bacteria  are  prominent  and  molds  absent 
or  relatively  few  in  numbers.  But  where  dextrose  is  added  to  the 
decomposing  proteins  molds  soon  appear  in  large  numbers.  There 
may  also  be  cited,  in  this  connection,  the  observation  of  Hilgard  that 
humus  should  contain  at  least  4  per  cent  of  nitrogen  if  it  is  to  furnish 
a  sufficient  quantity  of  available  nitrogen  compounds;  otherwise,  the 
soil  bacteria  seem  to  be  unable  to  decompose  it,  so  as  to  meet  the 
needs  of  the  growing  plants.  Many  similar  facts  could  be  cited  to 
show  that  as  a  culture  medium  the  soil  is  influenced  by  green  manures, 
barnyard  manure,  commercial  fertilizers,  lime,  tillage  and  any  other 
treatment  that  will  modify  the  quantity  as  well  as  the  quality  of  its 
organic  matter. 

THE  MINERAL  PORTION  OF  THE  SOIL. — The  moisture  films  sur- 
rounding the  soil  grains  contain  in  solution  substances  derived  from 
these  soil  grains.  A  particle  of  calcium  carbonate  will  be  surrounded 
by  a  moisture  film  containing  some  calcium  bicarbonate.  In  the 
same  way  particles  of  feldspar  may  give  rise  to  a  solution  of  potassium 
bicarbonate;  particles  of  apatite  to  a  solution  of  calcium  phosphate; 
particles  of  selenite  to  a  solution  of  calcium  sulphate;  particles  of 
protein  to  a  solution  of  ammonia,  etc.  In  view  of  the  fact  that  these 


MICROORGANISMS    AS    A   FACTOR    IN    SOIL   FERTILITY          359 

reactions  are  more  or  less  localized  and  diffusion  slow,  there  are,  un- 
doubtedly, in  the  soil  minute  zones  where  individual  species  are  more 
prominent  than  they  are  in  others.  For  example,  Heinze  has  found  it 
convenient  to  isolate  Azotobacter  by  inoculating  suitable  culture  solu- 
tions with  particles  of  calcium  carbonate  picked  out  from  the  soil. 
Evidently  these  organisms  were  present  in  much  greater  abundance 
on  these  particles  than  on  others  of  non-calcareous  origin.  Indeed, 
he  occasionally  obtained  in  this  manner  Azotobacter  membranes  that 
constituted  almost  pure  cultures.  The  more  general  significance  of 
this  relation  is  apparent  when  it  is  remembered  that  nitro-bacteria 
are  particularly  favored  by  magnesium  carbonate;  tubercle  bacteria 
by  gypsum  and  calcium  carbonate;  Azotobacter  by  calcium  phosphate 
and  calcium  carbonate;  photo-bacteria  by  sodium  chloride,  etc. 

Considerable  as  must  be  the  local  differences  in  any  one  soil,  they 
are  undoubtedly  even  more  pronounced  when  different  soils  are  com- 
pared. Extreme  conditions  are  met  with  in  certain  irrigated  soils 
in  which  a  marked  concentration  of  salts  occurs.  In  so  far  as  crop 
production  is  concerned,  it  is  stated  by  Hilgard  that  the,  upper  limit  is 
practically  reached  when  the  concentration  of  soluble  salts  in  the  irriga- 
tion water  is  about  4.55  g.  (70  gr.)  per  gallon.  Nevertheless,  in  Egypt 
and  the  Sahara  region  irrigation  water  is  occasionally  used  that  con- 
tains more  than  13  g.  (200  gr.)  of  soluble  salts  per  gallon.  Further 
differences  are  introduced  by  the  quality  of  these  salts,  e.g.,  the  pro- 
portion of  sodium  sulphate,  magnesium  sulphate,  sodium  chloride, 
sodium  carbonate,  etc.  Again,  instances  are  on  record,  as  in  the  investi- 
gations of  Headden  in  Colorado  and  California,  where  the  concentration 
of  nitrates  in  the  soil  water  is  so  great  as  to  kill  even  relatively  resistant 
plants  like  alfalfa.  It  is  to  be  shown  by  future  investigations  what  the 
effect  of  the  concentration  and  composition  of  such  salts  may  be  on  the 
soil  bacteria. 

In  humid  soils  conditions  are  less  extreme,  yet  even  here  the  variable 
concentration  and  composition  of  the  soil  solution  are  of  direct  moment 
for  the  different  microorganisms.  Granite  soils,  for  instance,  are  fairly 
well  supplied  with  phosphoric  acid  and  abundantly  with  potash,  but 
when  hornblende  is  lacking  they  are  apt  to  be  deficient  in  lime.  Ill- 
ventilated  clay  soils  may  contain  reduction  products  of  iron  salts,  while 
green  sand,  chalk,  slate,  shale,  sandstone  and  other  soils  may  have  their 
individual  peculiarities  from  the  standpoint  of  a  culture  medium. 


360  MICROBIOLOGY   OF   SOIL 

BIOLOGICAL  FACTORS 

MOLDS. — Distribution. — While  the  study  of  the  lower  bacteria  in  the 
soil  has  attracted  the  attention  of  many  investigators,  that  of  fungi  and 
actinomyces  received,  until  recently,  but  scant  consideration.  Fungi  oc- 
cur in  all  soils,  cultivated  as  well  as  uncultivated,  rich  or  poor  in  organic 
matter,  heavy  or  light  in  texture.  Most  of  them  are  obligate  sapro- 
phytes, although  facultative  parasites  are  found  in  large  numbers  in 
the  soil,  especially  where  single-cropping  or  short  rotations  favor  the 
survival  of  the  particular  organisms.  The  isolation  of  soil  fungi  has 
been  accomplished  either  by  the  dilution  method,  where  a  sample  of 
soil  was  shaken  with  water,  and  only  a  certain  dilution  was  used  for 
inoculation;  and  by  the  direct  method,  where  a  clump  of  soil  was  inocu- 
lated into  a  sterile  medium,  and  the  fungi  developing  on  it  were  isolated. 
About  1 50  different  species  of  fungi  have  been  isolated  from  different 
soils,  and  the  data  accumulated  by  investigators  in  this  country  and 
in  Europe  seem  to  point  to  the  fact  that  many  of  these  fungi  are 
universal  in  their  habitat,  since  the  same  species  are  recorded  to  have 
been  isolated  from  different  soil  types  and  in  different  localities.  Most 
of  the  work  done  refers  to  the  classification  of  the  organisms  isolated. 
The  largest  group  of  soil  fungi  belong  to  the  following  genera:  Mucor, 
Zygorrhynchus,  Rhizopus,  Aspergillus,  Penicillium,  Fusarium,  Tri- 
choderma,  Cephalosporium,  Monilia,  Cladosporium,  Alternaria,  and 
Acrostagmus.  Many  other  genera  have  been  isolated,  but  to  a  more 
limited  extent.  As  to  the  individual  species  occurring  in  the  soil, 
Hagem,  having  isolated  about  30  mucors  from  the  soil,  states  that 
certain  Aspergilli  occur  in  larger  numbers  than  all  the  mucors  taken 
together.  As  to  quantitative  relations,  no  exact  data  are  available. 
Some  investigators  report  only  several  hundred  fungi  per  g.  of  soil, 
while  others  record  as  many  as  1,000,000  per  g.  of  soil;  that  is  the 
total  number  of  spores  and  pieces  of  mycelium  that  develop  on  suitable 
media.  As  to  the  numbers  and  types  in  relation  to  depth,  Goddard 
concluded  that  there  does  not  seem  to  be  an  appreciable  variation  in 
numbers  at  the  different  soil  depths.  There  are  very  few  fungi  in  the 
soil  below  8  inches  and  one  of  the  most  common  forms  at  these  depths 
is  Zygorrhynchus  vuilleminii.  It  was  formerly  thought  that  soil  fungi 
are  abundant  only  in  acid  soils,  but  recent  investigations  make  it 
appear  that  also  limed  and  well-cultivated  soils  have  an  abundant 
fungus  flora. 


MICROORGANISMS   AS   A   FACTOR   IN   SOIL   FERTILITY          361 

The  plate  count  is  not  an  index  of  the  activity  of  the  molds  in  the 
soil,  but  merely  indicates  the  number  of  spores  present.  An  organism 
that  produces  a  large  number  of  spores,  particularly  when  these  resist 
drying,  will  be  found  by  the  plate  method  in  much  larger  numbers  than 
another  organism  which,  though  causing  a  greater  degree  of  chemical 
change  in  the  soil,  produces  fewer  and  less  resistant  spores.  A  method 
was  therefore  suggested  by  Waksman  which  would  permit  the  separa- 
tion of  molds  which  produce  mycelia  abundantly  and  readily  from 
those  that  develop  in  soils  only  under  special  conditions  of  moisture  and 
temperature.  The  method  consists  of  planting  a  lump  of  soil  into  the 
agar  in  a  Petri  dish  and  observing  the  development  of  the  mycelia  in 
the  soil.  It  is  obvious  that  the  mycelia  develop  more  readily  than 
the  spores  and  grow  out  into  the  agar.  In  this  manner  we  may  separate 
the  organisms  which  actually  live  in  the  soil.  Moreover,  the  fact  that 
the  same  species  of  molds  have  been  isolated  from  soils  in  different  parts 
of  the  world  would  tend  to  indicate  that,  when  conditions  become  favor- 
able, molds  vegetate  in  the  soil,  although  at  other  times  they  may  exist 
there  only  in  the  form  of  spores. 

Ammonification. — Miintz  and  Coudon,  and  after  them  Marchal, 
working  with  pure  cultures,  proved  conclusively  that  fungi  decompose 
organic  matter  and  cause  an  accumulation  of  ammonia  in  the  soil. 
Wilson  and  McLean  found  that  the  forms  of  MoniliasiYe  the  most  active 
ammonifiers  among  the  several  groups  of  organisms  studied,  while  the 
Aspergilli  showed  the  least  ammonifying  power.  More  recent  work 
has  confirmed  the  earlier  findings  and  has  proved  that  fungi  may 
play  an  active  part  in  the  decomposition  of  organic  matter,  and  the 
accumulation  of  ammonia. 

The  molds  have  been  shown  to  be  more  rapid  ammonifiers  than  the 
bacteria  and  actinomycetes.  Species  of  Trichoderma  have  been  found 
by  Waksman  and  Cook  to  transform  more  than  60  per  cent  of  the 
nitrogen  of  dried  blood  and  cottonseed  meal  into  ammonia  in  a  period 
of  seven  to  twelve  days.  This  comparatively  rapid  ammonia  produc- 
tion is  readily  explained  in  view  of  the  recent  information  on  the  energy 
requirements  of  microorganisms.  The  molds  decompose  organic 
matter  more  readily  than  do  the  actinomycetes  and  many  bacteria. 
They  consume  a  great  deal  more  energy  and  therefore  liberate  more 
nitrogen  as  a  waste  product  in  the  form  of  ammonia. 


362  MICROBIOLOGY   OF   SOIL 

Nitrogen-fixation. — Experiments  on  nitrogen-fixation  by  fungi  were 
carried  on  by  Jodin  as  early  as  1862.  He  observed  a  rich  fungus  growth 
on  nitrogen-free  media,  supplied  with  sugar,  tartaric  acid,  or  glycerin. 
Berthelot,  Saida,  Ternetz,  and  others  also  reported  fixation  of  atmos- 
pheric nitrogen  through  the  activities  of  fungi,  such  as  Aspergillus 
niger,  Alternaria  tennis  and  several  species  of  Monilia,  Penicillium, 
Mucorini  and  others.  But  other  investigators,  among  them  Wino- 
gradsky,  Czapek  and  Heinze,  were  unable  to  confirm  these  observa- 
tions. The  careful  work  of  Goddard  has  also  given  negative  results. 
Duggar  and  Davis,  eliminating  all  possible  errors  involved  in  this 
study,  could  not  demonstrate  any  nitrogen  fixation  for  Aspergillus 
niger,  Penicillium  digitatum,  Penicillium  expansum,  and  other  fungi, 
some  of  which  commonly  occur  in  the  soil.  Hence,  nitrogen  fixation 
by  soil  fungi  is  at  best  of  very  little  importance,  since  even  in  the  case 
of  positive  fixation  the  amounts  are  very  slight. 

Nitrogen  Utilization. — The  molds  assimilate  readily  available 
nitrogen  compounds  in  the  presence  of  available  carbohydrates.  In 
this  respect  they  may  readily  compete  with  higher  plants  in  using  up 
the  ammonia  and  nitrates  formed  in  the  soil  by  bacteria. 

Cellulose  Decomposition. — The  destruction  of  cellulose  in  the  soil  is 
due  to  a  large  extent,  to  the  activities  of  soil  fungi,  as  has  been  demon- 
strated by  several  investigators.  Cellulose  decomposition  by  fungi  was 
first  observed  in  the  study  of  plant  diseases.  Van  Iterson  used  filter 
paper  for  the  isolation  of  fungi,  by  exposing  this  medium  to  the  air  for 
twelve  hours.  Thirty-five  species  of  fungi  were  isolated  thus  proving 
that  a  large  number  of  cellulose-destroying  fungi  may  be  present  in 
the  air.  Appel  found  that  certain  species  of  Fusarium  destroyed  in 
fourteen  days  80  per  cent  of  the  filter  paper  used.  Marshall  Ward 
and  others  recorded  that  a  number  of  fungi  are  economically  impor- 
tant as  wood-destroyers.  Spores  of  a  pure  culture  of  Penicillium  sown 
on  sterile  blocks  of  spruce  wood,  germinated  and  grew  normally. 
Sections  of  the  wood  showed  that  the  hyphae  had  entered  the  starch- 
bearing  cells  of  the  medullary  rays  of  the  sapwood  and  consumed  the 
whole  of  the  starch.  MacBeth  and  Scales  found  that  when  the  medium 
is  slightly  alkaline,  certain  aerobic  bacteria  will  play  the  principal 
role  in  the  destruction  of  cellulose.  When  the  medium  is  acid,  molds 
and  higher  fungi  become  the  active  agents  of  destruction.  They  also 
found  that  the  cellulose-destroying  forms  multiply  with  great  rapidity 


MICROORGANISMS    AS    A   FACTOR   IN    SOIL   FERTILITY          363 

in  alkaline  soils  when  cellulose  in  the  form  of  filter  paper  is  added.  The 
power  to  destroy  cellulose  is  reported  for  a  number  of  species  of  Peni- 
cillia,  A  spergilli,  Trichoderma  and  other  organisms  which  belong  to  the 
common  soil  forms.  Though  the  fungi  may  play  an  important  part 
as  cellulose  destroyers  also  in  alkaline  soils,  in  acid  soils  where  the 
activity  of  bacteria  is  greatly  inhibited,  fungi  probably  play  a  pre- 
dominant role.  This  fact  led  Marshall  to  conclude  in  1893  that 
fungi  take  an  active  part  in  the  mineralization  of  the  organic  matter 
in  acid  humus  soils. 

Mycorrhiza. — Apart  from  the  so-called  soil  fungi,  there  exists  another 
group  known  as  mycorrhizal  fungi.  These  live  symbiotically  on  the 
roots  of  the  higher  plants.  Many  roots  of  forest  trees,  when  examined 
carefully,  show  that  there  is  a  union  between  the  mycelium  of  certain 
fungi,  usually  belonging  to  the  fleshy  fungi,  and  the  root  of  the  plant. 
This  union  is  called  a  "  mycorrhiza"  The  fine  filaments  of  the  fungus 
enter  the  cells  of  the  root.  These  organisms  were  thought  at  first 
to  supply  the  roots  with  water  and  soluble  plant  food  from  the  soil. 
The  power  to  fix  atmospheric  nitrogen  has  been  ascribed  to  these  organ- 
isms by  several  investigators.  But  aside  from  these  useful  so-called 
endotrophic  Mycorrhiza^  there  are  also  the  ectotrophic  Mycorrhiza 
which  probably  live  only  parasitically  upon  the  roots  of  plants. 

Actinomyces. — The  study  of  soil  Adinomyces  is  nearly  all  of  very 
recent  origin.  Several  years  ago  but  two  soil  Actinomyces  had  been 
definitely  described,  viz.,  Act.  albus  and  Act.  chromogenus.  The  work 
of  Krainsky,  of  Conn  and  of  Waksman  and  Curtis  has  demonstrated 
that  Actinomyces  are  widely  scattered  in  cultivated  soils.  The  last- 
named  investigators  have  shown  that  while  the  absolute  numbers  of 
Actinomyces  decrease  with  depth  of  soil,  their  relative  numbers  are 
materially  increased  so  that  if  at  a  depth  of  25  mm.  (i  inch)  there 
are  only  6  to  10  per  cent  of  Actinomyces  and  82  to  93  per  cent  of 
bacteria,  at  a  depth  of  750  mm.  (30  inches)  the  Actinomyces  form 
40  to  80  per  cent  of  the  total  microorganic  flora  of  the  soil.  The 
numbers  of  Actinomyces  in  the  surface  soil  vary  greatly  with  the  types 
of  soil  and  abundance  of  plant  food.  In  one  instance  1,300,000  A  cti- 
nomyces  were  found  in  a  total  of  15,000,000  bacteria  per  g.  of  rich 
meadow  soil.  The  actinomycetes  are  present  in  the  soil  both  in  the 
form  of  spores  and  vegetative  mycelium.  The  same  species  have  been 
isolated  from  North  America,  Canada,  Hawaiian  Islands  and  newly 


364  MICROBIOLOGY   OF   SOIL 

forming  soils  of  Tortugas  Island,  indicating  the  universal  occurrence 
of  these  organisms  in  the  soil.  Many  species  of  actinomycetes  have 
been  demonstrated  to  occur  in  the  soil  to  the  extent  of  millions  of  cells 
per  gram.  As  to  the  activities  of  Actinomyces  in  the  soil,  Beyer- 
inck  has  shown  that  the  Act.  chromogenus  produces  an  oxidizing  sub- 
stance, quinon  (C6H4O2)  which  may  play  an  important  part  in  the 
oxidation  of  organic  matter  in  the  soil.  Munter,  Krainsk'y  and  Scales 
have  demonstrated  that  many  Actinomyces  are  able  to  decompose  cellu- 
lose in  the  soil,  and  that  in  some  instances  this  ability  is  very  marked. 
Krainsky  records  that  soil  Actinomyces  need  very  little  nitrogen  for 
their  life  activities,  and  that  they  can  get  it  from  any  available  source. 
If  nitrates  are  present,  these  are  reduced  first  to  nitrites,  and'  then 
utilized.  Waksman  and  Curtis,  working  with  soil  sterilized  by  steam, 
did  not  find  any  great  accumulation  of  ammonia  through  the  activities 
of  Actinomyces,  although  different  species  seemed  to  show  marked  varia- 
tion in  their  power  to  accumulate  ammonia. 

ALG.E. — At  times  the  influence  of  algae  in  changing  the  character  of 
the  soil  as  a  culture  medium  for  bacteria  is  quite  considerable.  As 
chlorophyll-bearing  organisms  they  are  enabled  to  manufacture  sugar 
and  starch  with  the  aid  of  sunlight,  and  to  favor  thus  the  development 
of  A  zotobacter  and  of  other  microorganisms  dependent  for  their  energy 
on  the  organic  matter  in  the  soil.  Investigators  both  in  France  and 
in  Germany  have  found  that  the  fixation  of  nitrogen  in  sand  used  for 
pot  culture  experiments  occurs  in  the  surface  layer  possessing  a  growth 
of  alga?.  The  advocates  of  bare  fallows  attribute  the  greater  pro- 
ductivity of  fallowed  land  to  the  growth  of  algae,  the  accumulation  of 
nitrogen  through  their  influence  and  to  other  changes  affecting  the  soil 
bacteria. 

PROTOZOA. — It  has  been  known  for  a  long  time  that  certain  species 
of  protozoa  are  common  in  soils  and  that  their  food  consists  of  bacteria. 
To  what  extent  protozoa  play  a  part  in  soil  fertility  has  not  yet 
been  fully  explained,  even  though  Russell  and  Hutchinson  of  the 
Rothamsted  Experiment  Station  have  maintained  that  these  minute 
animals  are  extremely  important  in  that  they  maintain  a  certain  bac- 
terial equilibrium  in  the  soil.  Their  claim  is  mainly  based  on  the  fact 
that  partially  sterilized  soils  (either  by  means  of  heat  or  antiseptics) 
soon  come  to  contain  enormous  numbers  of  bacteria. 

It  is,  therefore,  assumed  by  them  that  this  abnormal  increase  is 


MICROORGANISMS   AS   A  FACTOR   IN   SOIL   FERTILITY          365 

made  possible  by  the  destruction  of  the  protozoa  (which  have  a  lower 
power  of  resistance  to  heat  and  antiseptics  than  bacteria)  that  normally 
check  the  increase  beyond  a  certain  point.  Under  the  conditions  re- 
corded a  causal  relationship  obtains  between  an  increase  in  numbers  of 
bacteria  and  the  rate  of  ammonia  production,  which  is  considered  to  be 
an  index  of  fertility. 

This  theory  has  been  the  basis  of  considerable  investigation,  much 
of  which  has  failed  to  corroborate  the  above  conclusions.  The  fact 
that  there  is  an  increase  in  bacterial  numbers  and  in  consequence, 
enhanced  fertility  of  the  soil  may  not  be  due  to  the  elimination  of 
protozoa  but  may  rather  be  ascribed  to  such  effects  of  the  partial 
sterilization  process  as  (i)  increase  in  available  food  for  bacteria; 
(2)  rendering  soil  toxins  insoluble;  (3)  destroying  bacterio- toxins; 
(4)  acceleration  of  the  biological  processes. 

It  has  even  been  noted  in  some  instances  that  partial  sterilization 
has  been  responsible  for  a  decrease  rather  than  increase  in  the  produc- 
tion of  ammonia.  Such  considerations,  among  others,  have  been  in- 
strumental in  stimulating  investigation  in  another  branch  of  soil  fertility, 
namely,  soil  protozoology.  There  has  been  difficulty  in  establishing 
suitable  methods  and  technic,  as  for  example  the  development  of 
media  favorable  for  the  isolation  and  culture  of  soil  protozoa,  although 
blood  meal  solution,  hay  infusion  and  soil  extract  have  been  used  to 
advantage.  The  organisms  have  been  counted  in  the  same  manner  as 
bacteria,  namely,  by  the  dilution  method,  or  by  means  of  a  standard 
platinum  loop.  An  adaptation  of  the  apparatus  used  in  the  counting 
of  blood  corpuscles  has  been  successfully  employed  by  Kopeloff, 
Lint  and  Coleman. 

A  study  of  the  morphology  and  life  history  of  soil  protozoa  reveals 
the  fact  that  encystment  occurs  under  most  conditions  which  are  not 
immediately  favorable,  as  for  example  slight  variations  in  moisture 
content,  or  food.  In  point  of  fact  this  period  of  the  protozoan  life  cycle 
which  is  analogous  to  the  spore-forming  stage  of  bacteria  forms  the 
basis  for  the  question  which  arises  as  to  the  existence  of  protozoa,  in 
their  trophic  form,  in  field  soils.  Of  the  well-defined  groups  of  pro- 
tozoa (page  14),  namely,  flagellates,  ciliates  and  amoebae,  many  types 
have  been  described.  Among  those  occurring  frequently  are:  Colpoda 
cucullus,  Boda  ovatus,  Colpidium  colpoda,  Amceba  terricola,  Monas,  etc. 
The  requirements  for  maximum  development  in  the  soil  for  these  organ- 


366  MICROBIOLOGY   OF   SOIL 

isms  are:  (i)  A  high  degree  of  moisture,  closely  approximating  saturation; 
(2)  an  abundant  supply  of  organic  matter;  (3)  moderate  temperature. 
The  thermal  death  point  of  active  forms  has  been  found  by  Goodey 
to  be  40°  to  50°,  and  for  the  cyst  forms  of  the  same  organisms  about 
72°.  The  optimum  temperature  for  most  forms  is  about  22°. 
Encystment  of  protozoa  occurs  within  wide  limits  in  an  alkaline  medium 
containing  up  to  .18  per  cent  NaOH,  and  in  the  presence  of  an  acidity 
represented  by  .09  per  cent  HC1. 

Protozoa  are  found  in  many  greenhouse  soils,  due  no  doubt  to  the 
fact  that  they  contain  a  high  degree  of  moisture  and  organic  matter. 
However,  in  dealing  with  field  soils  some  investigators  have  failed  to 
isolate  active  forms  of  protozoa,  whereas  others  record  the  presence  of 
large  numbers  of  these  organisms.  Their  distribution  appears  to 
parallel  that  of  bacteria,  namely,  the  greatest  number  of  protozoa  occurs 
within  the  upper  100  mm.  (4  inches)  of  soil,  with  a  decrease  down  to 
300  mm.  (12  inches),  which  represents  the  lower  limit  of  their  activity. 

As  regards  the  occurrence  of  the  various  groups  of  soil  protozoa, 
flagellates  are  found  to  be  dominant  over  ciliates  and  amoebae.  G.  P. 
Koch  has  found  that  the  development  of  soil  protozoa  in  artificial 
culture  solutions  varies  (i)  with  the  kind  of  media  employed;  (2)  the 
quantity  of  soil  used  for  inoculation;  (3)  drying  of  the  soil;  (4)  different 
kinds  of  soil  and  different  soils  of  the  same  kind;  (5)  the  temperature 
of  incubation. 

While  it  is  generally  accepted  that  protozoa  feed  upon  bacteria, 
until  the  relation  that  obtains  between  the  various  types  of  protozoa 
and  the  different  species  of  soil  bacteria  has  been  more  fully  investigated 
the  direct  effect  of  protozoa  upon  bacteria  must  remain,  to  a  degree, 
indeterminate. 

Soil  sterilization  has  had  a  practical  application  in  eliminating 
various  diseases  in  greenhouses  and  infested  fields.  Partial  steriliza- 
tion as  employed  by  Russell  and  Hutchinson  while  not  so  drastic, 
involves  serious  changes  in  the  soil,  which  might  be  considered  in  much 
the  same  light  as  the  phenomena  attending  complete  sterilization 
by  means  of  heat  and  antiseptics.  It  is  an  established  fact  that 
sterilization  is  responsible  for  increased  plant  growth,  and  to  explain 
this  phenomenon  the  following  theories  have  been  advanced: 

i.  R.  Koch's  theory  of  direct  stimulation  to  plant  growth — a 
physiological  effect  of  the  sterilizing  agency. 


MICROORGANISMS   AS   A   FACTOR   IN   SOIL   FERTILITY          367 

2.  Hiltner  and  Stormer's  theory  of  indirect  stimulation — an  altera- 
tion of  the  bacteriological  equilibrium  resulting  in  a  marked  develop- 
ment of  numbers  after  decimation. 

3.  Liebscher's  view  that  soil  sterilization  may  be"  regarded  in  the 
same  light  as  a  nitrogenous  fertilizer. 

4.  Russell  and  Hutchinson's  protozoan  theory  of  soil  fertility. 

5.  Pickering  and   Schreiner's   contention   that   the  alteration   in 
chemical  composition  is  largely  responsible  for  increased  plant  growth. 

6.  Greig-Smith  and  others  adhering  to  the  bacterio-toxin  hypothesis. 
HIGHER   PLANTS. — Higher  plants   modify   the   soil   as  a  culture 

medium  for  bacteria  in  at  least  three  ways.  The  root-hairs  come 
into  contact  with  the  moisture  films  surrounding  the  soil  grains  and 
not  only  modify  the  composition  of  the  film  water,  by  withdrawing  a 
portion  of  the  dissolved  matter,  but  also  change  its  character  by  secre- 
tions from  the  roots.  The  changes  thus  effected  must,  necessarily, 
modify  the  character  of  the  soil  and  the  soil  solution  as  a  culture 
medium.  Again,  the  rapid  removal  of  water  from  the  soil  by  growing 
crops  causes  the  film  water  to  become  more  concentrated  in  so  far,  at 
least,  as  some  salts  are  concerned.  Modifications  are,  also,  introduced 
thereby  in  the  proportions  of  oxygen,  nitrogen  and  carbon  dioxide  in 
the  soil  air.  Finally,  higher  plants  modify  the  soil  environment  for 
bacteria  by  their  root  and  stubble  residues.  For  example,  residues  of 
leguminous  plants,  being  richer  in  nitrogen  and  possessing  a  narrower 
carbon-nitrogen  ratio  than  the  corresponding  residues  of  non-legumes, 
will  affect  the  soil  somewhat  differently  than  the  latter. 

BACTERIA. — Occupying,  as  they  do,  the  leading  role,  bacteria 
demand  a  more  detailed  consideration;  in  fact,  most  of  the  biological 
discussions  of  soil  are  based  upon  a  knowledge  of  these  organisms. 

Numbers  and  Distribution  (Bacteria  in  Productive  and  Unproductive 
Soils). — The  numbers  of  bacteria  in  soils  well  supplied  with  organic 
matter  usually  range  from  3,000,000  to  200,000,000  per  g.,  as  shown 
by  the  agar  plate  method;  the  microscopic  count  will  show  as  high  as 
900,000,000  per  g.  of  soil.  These  numbers  vary  from  soil  to  soil,  and 
from  season  to  season  for  any  particular  soil.  The  numbers  of  fungi 
are  also  variable  and  may  reach  a  total  of  1,000,000  per  g.,  although 
it  still  remains  to  be  demonstrated  whether  the  large  numbers  thus 
found  represent  organisms  which  lead  an  active  life  in  the  soil  or  only 
spores  of  fungi  brought  in  by  external  agencies.  The  numbers  of 


368  MICROBIOLOGY   OF   SOIL 

Actinomyces  may  reach  1,000,000  or  more  per  g.  of  soil.  The  fungi 
almost  disappear  below  20  to  30  cm.,  while  the  actinomyces  do  not 
decrease  rapidly  at  depths  lower  than  30  cm. 

Distribution  dt  Different  Depths. — Most  of  the  soil  bacteria  are  found 
in  the  stratum  in  which  the  organic  residues  are  concentrated,  that  is, 
in  the  surface  soil.  Immediately  at  the  surface  the  rapid  evaporation 
and  the  germicidal  effect  of  direct  sunshine  act  as  disturbing  factors, 
hence  the  numbers  in  the  uppermost  25  to  50  mm.  (i  to  2  inches). are 
smaller  than  in  the  layer  of  soil  immediately  below.  Beyond  the 
depth  of  20  cm.  or  22  cm.  (8  or  9  inches)  the  numbers  diminish  rapidly. 
Material  from  a  depth  of  .6  m.  to  .9  m.  (2  to  3  feet)  is  nearly  sterile  in 
humid  regions.  Differences  occur,  however,  in  keeping  with  the 
mechanical  composition  of  the  soil.  In  light,  open  soils  the  bacteria 
are  not  only  carried  down  to  greater  depths  by  the  percolating  water, 
but  can  also  multiply  there,  thanks  to  better  aeration.  On  the  con- 
trary, fine-grained  compact  soils  are  more  effective  in  holding  back 
suspended  material  and  do  not  allow  the  bacteria  to  pass  downward  us 
readily.  Moreover,  the  less  thorough  aeration  of  these  soils  and  the 
accumulation  of  toxic  reduction  products  in  the  subsoil  serve  as  an 
effective  check  in  the  increase  of  bacteria  in  the  deeper  layers. 

In  irrigated  soils  of  the  arid  and  semi-arid  regions  bacteria  are  dis- 
tributed at  much  greater  depths.  Their  occurrence  2  m.  to  3  m. 
(8  or  10  feet)  below  the  surface  is  made  possible  not  only  by  the  better 
aeration  of  these  soils,  but  by  the  penetration  of  roots  to  great  depths 
and  the  accumulation  there  of  considerable  amounts  of  organic  matter. 
The  practical  significance  of  distribution  appears,  among  other  things, 
in  the  use  of  soil  for  inoculation  purposes;  for  instance,  it  is  reported  by 
Salstrom  that  in  making  peat  soils  arable  the  addition  of  small  amounts 
of  fertile  loam  increases  to  a  very  marked  extent  their  crop-producing 
power.  The  efficiency  of  the  inoculating  material  decreases  as  it  is 
taken  from  the  deeper  soil  layers.  Similarly,  in  the  use  of  alfalfa  soil 
for  the  inoculation  of  new  fields  the  most  efficient  material  is  secured  at 
a  depth  between  7.62  cm.  and  17.78  cm.  (3  and  7  inches). 

Seasonal  Variations  of  Bacterial  Numbers  and  Activities. — Conn  has 
reported  an  apparent  increase  of  bacteria  in  frozen  soil.  This  increase 
seems  to  be  due  to  an  actual  multiplication  of  the  organisms  rather  than 
to  a  mere  lifting  of  the  bacteria  from  lower  depths  by  capillary  action. 
The  greatest  increase  was  found  to  occur  during  the  winter  in  the  slow- 


MICROORGANISMS   AS   A   FACTOR   IN   SOIL   FERTILITY         369 

growing  bacteria  and  not  in  those  that  liquefy  gelatin  rapidly  or  in  the 
Actinomyces.  Conn  tries  to  account  for  the  phenomenon  by  assuming 
the  existence  of  two  groups  of  bacteria,  winter  and  summer  bacteria. 
The  latter,  he  thinks,  prevents  the  former  from  multiplying  rapidly 
in  warm  weather.  Hence,  the  increase  in  frozen  soils  is  not  to  be 
ascribed  directly  to  the  low  temperature,  but  to  the  depressing  effect 
of  the  cold  upon  the  summer  bacteria.  Brown  found  that  the  soil 
bacteria  diminish  during  the  fall  season  with  the  lowering  of  the 
temperature,  but,  when  the  soil  is  frozen,  an  increase  in  numbers 
occurs.  He  also  found  frozen  soils  to  possess  a  much  greater  ammoni- 
fying, denitrifying  and  nitrogen-fixing  power  than  non-frozen  soils. 
According  to  him,  the  lowering  of  the  freezing-point  of  the  capillary 
water,  due  in  part  to  the  concentration  of  salts  at  the  time  of  freezing, 
may  account  for  the  abnormal  bacterial  activities. 

Vass  recently  pointed  out  that  the  apparent  increase  of  bacteria  in 
frozen  soils  is  due  to  the  breaking  up  of  the  clumps  of  cells  rather  than 
to  growth  and  multiplication.  The  bacterial  activities  are  influenced 
by  freezing  only  in  so  far  as  it  affects  the  physical  properties  of  the  soil. 

Morphological  and  Physiological  Groups  (Morphological  Groups). — 
Rod-shaped  organisms  are  numerically  the  most  prominent  among  soil 
bacteria.  They  occur  at  times  to  the  extent  of  80  or  90  per  cent  of 
the  total  number.  Spherical  organisms  usually  constitute  less  than 
25  per  cent  of  the  bacterial  flora.  Spirilla  and  sarcinae  are  present  in 
slight  numbers.  Conditions  may  occur,  however,  when  the  proportion 
of  spherical  organisms  is  markedly  increased.  This  happens,  par- 
ticularly, when  large  quantities  of  composted  manure  (rich  in  spherical 
organisms)  is  added  to  the  soil. 

Conn  has  shown  that  non-spore-forming  bacteria  (mostly  immotile 
rods)  are  the  most  abundant  of  all  soil  microorganisms.  Next  to  them 
in  abundance  are  the  various  types  of  Actinomyces,  referred  to  elsewhere 
in  this  book.  Spore-forming  bacteria  are  also  quite  common,  but  are 
apparently  of  no  great  importance  in  normal  soil.  Among  the  most 
prominent  soil  bacteria  are  non-spore-forming,  slowly  liquefying  or 
non-liquefying,  short  rods;  rapidly  liquefying,  non-spore-forming,  short 
rods  with  polar  flagella  represented  by  Ps.  fluorescens;  spore  formers, 
which  seem  to  come  from  spores  instead  of  from  active  organisms.  A 
few  micrococci  and  members  of  the  B.  radicicola  group  have  been 
demonstrated. 

24 


370  MICROBIOLOGY   OF   SOIL 

Among  the  rod-shaped  species  B.  mycoides,  B.  subtilis,  B.  mesen- 
teriCus,  B.  tumescens  and  other  members  of  the  subtilis  group  are  quite 
prominent.  Members  of  the  amylobacter  group  are  seldom  absent. 
Members  of  the  proteus  group  and  various  fluorescens  are  always 
present,  while  Bad.  cerogenes  and  allied  species  are  common  inhabitants 
of  the  soil. 

(Physiological  Groups). — In  the  decomposition  of  organic  matter  in 
the  soil  certain  important  changes  in  both  nitrogenous  and  non-nitro- 
genous material  are  accomplished  by  definite  groups  of  bacteria.  The 
breaking  down  of  protein  substances  is  accomplished  by  the  forma- 
tion of  ammonia,  nitrites  and  nitrates.  These  in  turn  may  be  trans- 
formed back  into  more  complex  amino-compounds,  peptones,  and  pro- 
teins, or  they  may  be  destroyed  with  the  evolution  of  free  nitrogen. 
Moreover,  there  are  groups  of  bacteria  capable  of  joining  non-nitro- 
genous organic  matter  to  elementary  nitrogen  and  of  producing  thus 
nitrogen  compounds.  Again,  there  are  groups  of  bacteria  bearing 
distinct  and  important  relations  to  the  decomposition  of  cellulose,  or 
the  transformation  of  its  cleavage  products,  methane  and  hydrogen. 
There  are,  likewise,  definite  groups  of  bacteria  concerned  in  the 
transformation  of  sulphur  and  its  compounds,  and  of  ferrous  compounds. 

METHODS  or  STUDY 

METHODS  FOR  COUNTING  BACTERIA. — There  are  two  methods  for 
the  quantitative  determination  of  bacteria  in  the  soil:  the  plate  method 
and  the  direct  count  method.  By  the  use  of  the  plate  method  we  can 
obtain  only  relative  results,  since  not  all  soil  bacteria  are  able  to  grow 
and  develop  into  colonies  even  on  the  most  suitable  media.  The  plate 
method  shows  cells  of  bacteria  that  are  able  to  develop  under  laboratory 
conditions  but  furnishes  no  direct  evidence  as  to  their  exact  number. 
Conn  therefore  suggested  the  direct  count  method,  already  employed  suc- 
cessfully in  the  bacteriological  examination  of  milk.  A  smear  is  prepared 
by  spreading  o.i  c.c.  of  the  soil  infusion  over  an  area  of  i  sq.  cm.,  then 
stained  with  Rose  Bengal  in  carbolic  acid.  The  bacteria  are  colored 
deep  pink  or  red,  while  the  mineral  particles  remain  uncolored  and  most 
of  the  organic  matter  is  unstained  or  stained  yellow  or  light  pink. 
The  bacteria  are  then  counted  by  means  of  an  oil-immersion  objective 
and  a  high  power  eye-piece.  The  actual  numbers  of  bacteria  detected 


MICROORGANISMS    AS    A    FACTOR   IN    SOIL   FERTILITY          371 

by  the  microscope  is  probably,  according  to  Conn,  5,  10  or  over  20  times 
greater  than  that  indicated  by  the  plate  method.  The  discrepancy  is 
due  to  the  failure  of  many  cells  to  produce  colonies  rather  than  to  the 
occurrence  of  large  clumps  that  o  not  break  up  in  the  process  of 
plating. 

QUANTITATIVE  RELATIONS. — Since  the  early  work  of  Koch  in  1881 
many  investigators  have  determined  the  number  of  bacteria  in  soil 
samples,  by  means  of  the  plate  method.  It  is  well  known,  however, 
that  on  ordinary  gelatin  or  agar  plates  kept  under  aerobic  conditions 
but  a  fraction  of  the  soil  organisms  produce  visible  colonies.  The 
anaerobic  species  do  not  appear,  nor  do  aerobic  Azotobacter,  and  nitro- 
bacteria,  while  other  common  soil  organisms  form  colonies  sparingly 
or  not  at  all.  .  By  employing  synthetic  agar  media  instead  of  beef  broth 
gelatin  or  agar,  Lipman  and  Brown  have  succeeded  in  securing  the 
growth  of  a  much  larger  number  of  colonies  from  any  given  quantity 
of  soil,  yet  even  these  larger  numbers  were  incomplete  for  reasons 
mentioned  above. 

H.  Fischer  recommends  a  simple  medium  of  agar  to  which  nothing 
has  been  added  but  soil  extract  (prepared  by  extracting  with  a  .1 
per  cent  solution  of  Na2CO3)  and  potassium  phosphate.  Following 
the  path  of  Lipman  and  Brown  in  reducing  the  content  of  organic 
matter,  Temple  employed  i  g.  of  peptone  per  1.  as  a  culture  medium 
and  obtained  satisfactory  results.  Brown  has  further  modified  the 
formula  of  Lipman  and  Brown  by  replacing  the  .05  g.  of  peptone  with 
.1  g.  of  albumin,  and  obtained  results  which  were  somewhat  superior. 
In  a  comparison  of  culture  media,  Conn  considers  the  former  media 
undesirable  for  quantitative  purposes  because  they  contain  substances 
of  indefinite  chemical  composition,  and  offers  an  agar  medium  con- 
taining no  organic  matter  except  agar,  dextrose  and  sodium  asparag- 
inate,  and  also  a  soil-extract  gelatin  which  is  valuable  for  qualitative 
purposes.  Other  media  that  have  been  suggested,  after  a  comparison 
of  all  of  the  above-mentioned  media,  are  the  urea-ammonium  nitrate 
agar  of  R.  C.  Cook  and  the  tap  water  gelatine  and  asparaginate  agar  of 
Conn.  It  is  evident,  therefore,  that  the  results  secured  in  the  counting 
of  soil  bacteria  have  only  a  relative  value.  With  the  same  media  and 
methods  some  information  may  be  secured  concerning  the  influence 
of  fertilization,  tillage,  liming,  etc.,  on  certain  of  the  soil  bacteria.  But 
even  this  information  must  be  properly  discounted,  since  equal  numbers. 


372  MICROBIOLOGY   OF   SOIL 

do  not  necessarily  mean  equal  amounts  of  chemical  work  accomplished ; 
for  example,  there  is  no  certainty  that  1,000,000  of  decay  bacteria 
derived  from  one  soil  will  accomplish  exactly  as  much  decomposition 
as  the  same  number  of  similar  organisms  from  another  soil.  Otherwise 
stated,  individual  cells  differ  in  their  physiological  efficiency  from  other 
cells  of  the  same  species. 

QUALITATIVE  REACTIONS. — By  modifying  the  composition  of  the 
culture  media  different  physiological  groups  may  be  favored  in  their 
development.  In  this  manner  the  silica  jelly  medium  proposed  by 
Winogradski,  or  the  gypsum  plates  proposed  by  Omelianski  may  be  em- 
ployed for  making  numerical  comparisons  of  nitro- bacteria  in  different 
soils.  In  like  manner  Beijerinck's  mannit  agar  may  be  used  for  the 
numerical  comparison  of  Azotobacter,  and  other  media  can  be  adapted 
for  the  quantitative-qualitative  determination  of  urea,  denitrifying, 
methane,  and  still  other  physiological  groups  of  microorganisms, 
modified  Czapek's  agar  and  Krainsky's  agar  can  be  used  for  actino- 
mycetes  and  raisin  agar  for  molds. 

There  is  no  doubt  that  the  quantitative-qualitative  method  just  out- 
lined may  be  made  to  yield  valuable  information.  Yet  it,  too,  possesses 
defects  already  noted  in  connection  with  the  more  strictly  quantitative 
method.  Apart  from  the  vast  amount  of  work  involved  in  the  prepa- 
ration of  a  large  number  of  media  and  in  the  counting  of  colonies  on 
many  plates,  this  method  fails  to  indicate  differences  in  physiological 
efficiency.  Furthermore,  the  colonies  of  the  specific  organisms  sought 
are  almost  invariably  accompanied  by  those  of  foreign  species  not 
always  easily  distinguished.  With  these  limitations  properly  recognized 
and  with  further  improvement  in  the  constitution  of  special  media  the 
method  may  be  made  useful  in  supplementing  data  secured  by  other 
methods. 

TRANSFORMATION  REACTIONS. — Instead  of  counting  soil  bacteria  in 
accordance  with  colonies  produced  in  general  or  special  media,  soil 
investigators  have  attempted  to  measure  the  bacteriological  functions  of 
soils  by  comparing  more  or  less  definite  quantities  of  the  latter  under 
known  conditions.  This  method  was  employed  by  Wollny  and  others 
in  studying  the  factors  that  affect  the  formation  of  carbon  dioxide  in 
soils.  It  was  also  used  by  Schloesing  and  Muntz  and  their  followers  in 
similar  studies  on  nitrate  formation.  A  method  somewhat  similar  in 
principle  but  different  in  its  application  was  proposed  by  Remy  in 


MICROORGANISMS   AS    A    FACTOR   IN    SOIL   FERTILITY          373 

1902.  He  suggested  the  use  of  special  media  for  the  quantitative 
estimation  of  different  physiological  reactions;  thus,  making  a  i  per 
cent  solution  of  peptone  and  inoculating  with  equivalent  quantities  of 
soil,  he  caused  the  decomposition  of  the  peptone  and  the  formation  of 
ammonia,  and  secured  comparisons  of  the  ammonifying  power  of 
different  soils.  In  a  similar  manner  he  used  special  solutions  for  com- 
paring quantitatively  the  transformation  accomplished  by  nitrifying, 
denitrifying  or  nitrogen-fixing  bacteria. 

Remy's  method  has  been  extensively  tested  by  Lohnis,  Ehrenberg, 
Lipman  and  others.  It  has  been  shown  to  possess  a  serious  defect  in 
that  it  deals  with  conditions  unlike  those  occurring  in  the  soil  itself. 
For  this  reason  more  recent  investigations  have  been  carried  on  in 
weighed  portions  of  soil  rather  than  in  culture  solutions  inoculated  with 
10  per  cent  of  soil  as  is  done  in  Remy's  method. 

RATE  OF  OXIDATION  OF  CARBON. — The  rate  of  decomposition  of 
humus  or  of  other  organic  matter  in  the  soil  may  be  measured,  as  was 
done  by  Wollny,  by  determining  the  amount  of  carbon  dioxide  evolved 
in  weighed  quantities  of  material  kept  under  definite  conditions.  The 
influence  of  temperature,  moisture,  aeration,  organic  matter,  anti- 
septics, etc.,  has  been  determined  in  this  manner.  The  same  method 
may  be  used  in  studying  decay,  and  factors  influencing  decay,  in  soils 
in  the  field. 

More  recently  Russell  and  his  associates  have  modified  the  method 
in  that  they  have  determined  the  rate  of  oxidation  of  carbon  not  by 
measuring  the  carbon  dioxide  evolved,  but  by  estimating  the  amount  of 
oxygen  absorbed.  In  either  case  decay  is  measured  from  the  carbon 
standpoint.  The  method  based  on  this  principle  should  find  wide 
application  in  future  soil  fertility  investigations. 

Potter  and  Synder  measured  the  amount  of  carbon  dioxide  evolved 
from  sterilized  soil  when  inoculated  with  soil  emulsion  or  with  cultures 
of  molds.  The  latter  produced  in  nearly  all  cases  as  much  carbon  diox- 
ide as  the  soil  suspension  and  in  some  cases  more.  This  fact  led  them 
to  suggest  that  molds  are  probably  active  in  normal  soils.  Gainey 
pointed  out  that  there  is  a  similarity  and  agreement  between  the  curves 
representing  carbon  dioxide  and  ammonia  formation  in  soils.  The 
relative  content  and  availability  of  the  carbon  and  nitrogen  sources  in 
the  soil  influence  greatly  the  relative  amounts  of  carbon  dioxide  and 
ammonia  produced. 


374  MICROBIOLOGY   OF    SOIL 

RATE  OF  OXIDATION  OF  NITROGEN. — Another  method  or  series  of 
methods  for  studying  decomposition  processes  in  the  soil  may  be  based 
on  the  determination  of  nitrogen  compounds  formed  in  the  breaking 
down  of  proteins.  Two  of  the  derivatives  of  protein,  namely,  ammonia 
and  nitrate,  have  been  used  successfully  to  gauge  the  decomposition  of 
organic  matter  in  the  soil.  The  recent  results  secured  by  Lipman  and 
his  associates  demonstrate  that  ammonia  formation  from  dried  blood  in 
weighed  quantities  of  soil  may  serve  as  a  very  accurate  measure  of 
decay  from  the  nitrogen  standpoint.  Corresponding  determination  of 
nitrates  may  similarly  be  employed  in  tracing  protein  cleavage  and 
transformation  as  influenced  by  the  various  factors  of  season,  soil 
and  cultivation. 

ADDITION  OF  NITROGEN. — At  least  one  other  bacteriological  factor 
in  soils  should  be  mentioned  here  as  deserving  attention  in  a  systematic 
study  of  soil  fertility  from  the  nitrogen  standpoint.  It  is  known  that 
Azo-bacteria  are  widely  distributed  in  arable  soils,  and  that  they  are 
more  prominent  in  some  regions  than  they  are  in  others.  The  student 
of  soil  fertility  finds  it  desirable,  therefore,  to  study  azotofication  in 
different  soils,  and  employs  (for  this  purpose)  mannit  solutions  like 
those  proposed  by  Beyerinck,  sand  cultures  supplied  with  sugar  solu- 
tions like  those  proposed  by  Fischer,  or  weighed  quantities  of  soil  mixed 
with  sugar  as  suggested  by  Koch. 

The  methods  referred  to  above  make  possible  thus  the  study  of 
ammonification,  nitrification  and  azotofication  under  controlled  con- 
ditions and  permit,  thereby,  the  measure  of  bacteriological  factors  in 
soil  fertility  from  the  nitrogen  standpoint. 

REACTIONS  CONCERNING  CALCIUM,  MAGNESIUM,  SULPHUR  AND 
PHOSPHORUS. — In  addition  to  the  purely  chemical  methods  available  for 
the  study  of  these  constituents,  microbiological  methods  have  also  been 
suggested.  In  some  of  his  still  unpublished  experiments  with  Azoto- 
bacter  Lipman  employed  solutions  of  mannit  in  distilled  water,  provided 
with  small  quantities  of  sterile  soils  which  were  to  supply  the  organisms 
with  the  essential  mineral  constituents.  In  this  manner  interesting 
data  were  secured  on  the  availability  of  phosphorus  compounds  in 
different  soils;  similarly,  Christensen  has  suggested  the  use  of  Azoto- 
bacter  for  determining  the  lime  requirements  of  soils,  and  Butkevich 
has  experimented  with  cultures  of  Aspergillus  niger  in  determining  the 
availability  of  the  mineral  constituents. 


CHAPTER  II 
THE  DECOMPOSITION  OF  ORGANIC  MATTER  IN  THE  SOIL 

CARBOHYDRATES 

Origin. — The  sugars,  starches,  vegetable  gums  and  allied  pec  tine 
substances,  as  well  as  the  cellulose,  contained  in  roots  and  other  crop 
residues  add  large  quantities  of  carbohydrates  to  the  soil.  The  crop 
residues  are  augmented  still  further  by  green  manures  and  animal 
manures  whenever  these  are  used.  A  good  growth  of  timothy,  for 
example,  may  increase  the  content  of  organic  matter  in  the  surface 
soil  by  250-500  kg.  (500  or  1,000  pounds)  per  acre,  and  three-quarters 
of  this  consists  of  carbohydrates.  In  the  same  manner,  a  green  ma- 
nure crop,  or  an  application  of  barnyard  manure  may  add  to  the  land 
as  much  as  1,500  pounds,  or  even  more,  of  carbohydrates  per  acre. 
These  carbohydrates  contain  a  large  proportion  of  cellulose. 

The  Decomposition  of  Cellulose. — Pure  cellulose  (page  237), 
(C6HioO5)x  is  a  rather  inert  substance,  as  exemplified  by  the  resistance 
of  cotton  and  flax  fiber  to  decomposition  processes.  It  is  well  known, 
nevertheless,  that  even  cellulose  is  in  the  end  decomposed  and  resolved 
into  simple  compounds.  Plant  roots,  leaves  and  stems,  as  well  as  the 
trunks  of  fallen  trees,  gradually  disintegrate  and  vanish.  Under  favor- 
able conditions  this  may  proceed  rapidly,  as  is  indicated  by  the  process 
in  septic  tanks,  or  in  manure  heaps  on  the  one  hand,  and  in  open 
sandy  soils  on  the  other.  The  disappearance  of  cellulose  may  be  ac- 
complished by  (a)  anaerobic  organisms,  (b)  by  aerobic  organisms,  (c) 
by  denitrifying  bacteria,  (d)  by  molds  and  (e)  by  actinomycetes  usually 
classed  as  higher  bacteria. 

The  Production  of  Methane  and  Hydrogen. — The  decomposition 
of  pure  cellulose  and  the  formation  of  methane  and  hydrogen  mixed 
with  other  gases  was  first  noted  by  Popov  in  1875.  Some  years 
later  Tappeiner  and  also  Hoppe-Seyler  confirmed  Popov's  observa- 
tions that  nearly  pure  cellulose  in  the  form  of  Swedish  filter-paper,  or 
cotton  fiber  may  be  fermented  by  bacteria  with  the  evolution  of 
methane,  carbon  dioxide  and  occasionally  also  of  hydrogen.  These 

375 


376  MICROBIOLOGY   OF   SOIL 

investigators  ascribed  the  decomposition  of  cellulose  to  an  organism 
found  by  Trecul  in  decomposing  vegetable  materials,  and  named  by 
him  Amylobacter  in  1865,  because  of  the  blue  color  assumed  by  it  when 
stained  with  iodine. 

Subsequent  investigations  by  Omelianski  begun  in  1894  and  con- 
tinued through  a  period  of  years  demonstrated  the  presence  of  specific 
anaerobic  organisms  in  decomposing  cellulose.  He  described  two  dis- 
tinct species  of  long,  slender  bacilli,  assuming  the  clostridium  form  when 
in  the  spore  stage.  Morphologically  the  organisms  can  hardly  be  dis- 
tinguished, but  physiologically  they  show  important  differences  in  that 
one  causes  the  fermentation  of  cellulose  with  the  production  of  gases 
consisting  of  carbon  dioxide  and  methane,  while  the  gases  produced  by 
the  other  consist  of  carbon  dioxide  and  hydrogen; hence  the  one  is  desig- 
nated by  Omelianski  as  the  methane  bacillus  and  the  other  the  hydro- 
gen bacillus.  These  organisms  do  not  stain  blue  with  iodine,  and  do  not 
belong,  therefore,  to  the  butyric  bacilli  designated  as  Amylobacter  by 
earlier  investigators.  Omelianski's  investigations  make  it  appear  that 
the  butyric  organisms  are  not  capable  of  fermenting  cellulose  proper. 

In  culture  solutions  containing  mineral  salts  and  nitrogen  in  the  form 
of  ammonium  compounds  the  decomposition  of  filter-paper  and  other 
forms  of  cellulose  proceeds  with  considerable  rapidity.  Calcium  car- 
bonate must  be  added  to  neutralize  the  acids  formed,  otherwise  the 
fermentation  soon  comes  to  a  standstill.  In  one  of  Omelianski's  experi- 
ments begun  in  October,  1895,  and  ended  in  November,  1896,  3.3471 
g.  of  cellulose  was  decomposed  by  a  nearly  pure  culture  of  hydrogen 
bacilli.  The  products  consisted  of  2.2402  g.  fatty  acids,  .9722  g.  carbon 
dioxide  and  .0138  g.  of  hydrogen,  a  total  of  3.2262  g.  which  nearly 
accounts  for  all  of  the  cellulose  destroyed.  The  fatty  acids  were  made 
up  of  butyric  and  acetic  acids  with  a  slight  proportion  of  some  higher 
homologue,  probably  valerianic  acid. 

In  a  similar  experiment  with  an  apparently  pure  culture  of  the 
methane  bacillus,  begun  in  December,  1900,  and  ended  in  April,  1901, 
fermentation  began  after  an  incubation  period  of  about  one  month,  and 
the  entire  volume  of  gas  gradually  evolved  was  552.2  c.c.  This  mix- 
ture consisted  of  190.8  c.c.  methane  and  361.4  c.c.  carbon  dioxide.  The 
products  formed  from  the  2.0065  g-  cellulose  consumed  included 
1.0223  g.  fatty  acids,  .8678  g.  carbon  dioxide  and  .1372  g.  of  methane, 
or  a  total  of  2.0273  g.  The  slight  difference  in  weight  in  favor  of  the 


DECOMPOSITION   OF   ORGANIC   MATTER   IN   THE    SOIL          377 

fermentation  products  falls  within  the  limit  of  error.  These  experi- 
ments show  that  about  one-half  of  the  fermentation  products  is 
gaseous  and  that  the  other  half  consists  of  acetic  and  butyric  acids. 

McBeth  has  shown  that  the  cellulose-dissolving  bacteria  are  unable 
to  produce  gaseous  products  in  cellulose  or  sugar  solutions  in  which 
they  make  a  luxuriant  growth.  The  compounds  formed,  under  natural 
conditions,  by  the  cellulose  dissolving  bacteria  are  used  by  other  micro- 
organisms and  split  into  simpler  products.  The  carbon  dioxide  formed 
is  presumably  due  in  all  cases  to  secondary  fermentations.  The 
organic  acids  noted  by  early  investigators  were,  for  the  most  part  at 
least,  presumably  due  to  secondary  fermentations  and  not  to  the  action 
of  the  cellulose-dissolving  forms. 

The  Oxidation  of  Methane,  Hydrogen  and  Carbon  Monoxide. — Aside 
from  cellulose,  methane  may  also  be  produced  from  various  other  carbo- 
hydrates, organic  acids  and  proteins.  Large  amounts  of  methane  are 
thus  contributed  to  the  atmosphere  by  swamps,  manure  heaps  and  low- 
lying  meadows.  In  a  purely  chemical  way  methane  may  also  be  set 
free  from  volcanoes  and  mineral  springs.  The  constant  additions  of 
methane,  ethane,  hydrogen  and  carbon  monoxide  represent  a  consid- 
erable amount  of  potential  energy.  It  is  important  to  know,  therefore, 
whether  these  materials  are  at  all  utilized. 

That  methane  may  be  utilized  by  bacteria  as  a  source  of  energy  was 
first  shown  by  Sohngen  in  1905.  He  isolated  an  organism  named  by 
him  B.  methanicus  that  showed  itself  capable  of  growing  in  inorganic 
solutions  confined  over  an  atmosphere  of  methane,  oxygen  and  nitrogen. 
The  methane  gradually  disappeared  and  there  were  formed  considerable 
quantities  of  organic  matter.  The  ability  to  oxidize  methane  has  been 
claimed  for  a  number  of  other  organisms  by  Sohngen  and  others. 

Early  observations  on  the  ability  of  moist  soil  to  cause  the  oxidation 
of  hydrogen  are  credited  to  de  Saussure  (1838).  Many  years  later 
(1892)  Immendorff  called  attention  to  the  same  fact.  It  was  not, 
however,  until  1905  that  the  oxidation  of  hydrogen  was  shown  to  be  a 
specific  biological  process.  In  that  year  papers  by  Sohngen  and  Kaserer 
reported  experiments  wherein  inorganic  solutions  confined  under  an 
atmosphere  of  hydrogen,  oxygen  and  carbon  dioxide  and  inoculated  with 
very  small  quantities  of  soil  developed  a  bacterial  membrane  at  the 
surface.  The  hydrogen  was  oxidized  and  organic  matter  produced  at 
the  expense  of  the  energy  set  free.  The  observations  just  noted  have 


MICROBIOLOGY   OF   SOIL 

been  confirmed  by  other  investigators,  by  means  of  mixtures  and  single 
species  of  soil  bacteria.  Finally  it  should  be  added  here  that  B. 
oligocarbophilus  previously  isolated  by  Beijerinck  and  Van  Delden  is 
able,  according  to  Kaserer,  to  oxidize  also  carbon  monoxide. 

The  Cleavage  and  Fermentation  of  Sugars,  Starches  and  Gums. — 
Sugars  (page  233)  are  a  very  acceptable  source  of  food  and  energy 
for  soil  bacteria.  A  culture  solution  containing  suitable  mineral 
salts  and  sugar  ferments  readily  when  inoculated  with  a  small  amount 
of  fresh  soil.  When  no  combined  nitrogen  is  added,  Azotobacter,  or  B. 
(Clostridium)  pasteurianus  (or  both),  may  come  to  the  fore.  The  cleav- 
age products  then  include  alcohols,  organic  acids  and  carbon  dioxide. 
With  B.  (Clostridium)  pasteurianus  butyric  acid  is  one  of  the  prominent 
cleavage  products.  When  combined  nitrogen  is  also  added  to  the 
culture  solution  other  organisms  will  develop  prominently,  notably 
members  of  the  subtilis  group,  butyric  bacteria,  aerogenes,  etc.  In  the 
soil  itself  the  addition  of  sugar  leads  to  a  very  marked  increase  in 
number  and,  if  acid  production  is  favored,  molds  may  subsequently 
become  prominent.  In  general  it  may  be  said  that  butyric,  propionic, 
acetic,  formic  and  lactic  acid,  and  ethyl,  propyl,  butyl  and  iso-butyl 
alcohol  are  common  cleavage  products. 

In  the  case  of  starch,  pectins  and  pentosans,  similar  conditions  hold 
good.  Diastatic  enzymes  seem  to  be  produced  by  various  bacteria 
as  well  as  by  molds  and  actinomycetes.  Members  of  the  subtilis  group 
and  B.  Huorescens  seem  to  be  able  to  transform  starch  into  sugar  with- 
out difficulty.  It  needs  hardly  be  added  here  that  the  vast  quantities 
of  organic  acids  and  of  carbon  dioxide  thus  formed  must  play  an  im- 
portant role  in  the  breaking  down  of  the  mineral  constituents  in  the 
soil. 

FATS  AND  WAXES 

Origin  and  Decomposition.— Plant  substances  contain  varying 
proportions  of  fats  and  waxy  materials.  In  the  dry  matter  of  grasses 
and  cereal  straw  crude  fat  is  usually  present  to  the  extent  of  1.5  to 
2.0  per  cent.  In  hay  made  from  clover  and  other  legumes  the  propor- 
tion of  crude  fat  is  rather  more  than  2  per  cent.  In  cereal  grains  it 
may  range  up  to  4  or  5  per  cent  while  in  soy  beans  the  content  of 
crude  fat  is  19  per  cent,  in  germ  oil  meal  22  per  cent  and  flax  seed 
meal  34  per  cent. 


DECOMPOSITION   OF   ORGANIC   MATTER   IN   THE    SOIL         379 

Under  the  influence  of  enzymes  produced  by  molds,  yeasts  and 
bacteria  the  fatty  acids  occurring  as  glycerides  are  decomposed  into 
glycerin  and  fatty  acids.  The  extent  of  fat  decomposition,  brought 
about  largely  by  molds  in  the  opinion  of  some,  is  shown  by  numerous 
experiments  with  peanut  cake,  olive  press  cake,  cottonseed  meal, 
almond  oil,  corn  meal,  etc.  In  a  number  of  these  experiments  Asper- 
gillus  niger  seemed  to  be  particularly  efficient  in  decomposing  fats. 
Analogous  decomposition  processes  may  occur  in  the  soil  as  proved  by 
the  experiments  of  Rubner. 

ORGANIC  ACIDS 

Source. — The  cleavage  products  of  proteins  include  large  quantities 
of  amino-acids.  The  latter  are  still  further  transformed  and  yield  a 
variety  of  fatty  acids.  The  carbohydrates  being  present  in  larger 
quantities  than  the  proteins  are  still  more  important  as  a  source  of 
organic  acids.  Finally,  the  fats,  gums,  and  higher  alcohols  contribute 
additional  quantities  of  the  latter.  Among  the  more  simple  acids, 
acetic,  propionic,  butyric,  succinic  and  lactic  are  common.  The  extent 
of  acid  production  was  already  indicated  in  connection  with  cellulose 
decomposition  by  the  methane  and  hydrogen  bacilli.  Apart  from  these 
organisms,  organic  acids  are  formed  by  nearly  every  important  species 
of  soil  bacteria;  moreover,  the  tissues  of  dead  plants  and  animals  are 
not  the  sole  source  of  organic  acids  in  the  soil.  According  to  Stoklasa 
conditions  may  occasionally  occur  in  the  latter,  especially  when 
atmospheric  oxygen  is  excluded,  that  favor  the  excretion  by  plant  roots 
of  appreciable  quantities  of  acetic  acid. 

Aside  from  the  organic  acids  produced  by  bacteria,  we  must  also 
consider  the  acids  produced  by  molds;  among  these  oxalic  and  citric 
acids  are  most  important.  Certain  members  of  the  Aspergillus  niger 
group  are  able  to  convert  as  much  as  40  per  cent  of  the  sugar  in  solution 
into  citric  acid ;'  the  latter  is  then  further  oxidized  into  oxalic  acid.  In 
addition  to  the  Aspergilli,  several  Penicillia,  Mucors,  Absidia  and  other 
molds,  which  have  been  isolated  from  the  soil,  are  able  to  produce  citric 
or  oxalic  acids,  or  both.  The  acid  produced  in  the  culture  medium  is 
either  allowed  to  accumulate  or  is  further  oxidized.  Aspergillus  niger 
oxidizes  sugar  first  into  citric  acid,  the  latter  is  then  oxidized  to  oxalic 
acid  and  finally  to  carbon  dioxide. 


380  MICROBIOLOGY   OF   SOIL 

Transformation  and  Accumulation. — Salts  of  organic  acids  are 
suitable  as  food  for  a  wide  range  of  soil  bacteria.  Azotobacter  will 
•  readily  make  use  of  acetates,  propionates  and  butyrates.  A  number  of 
denitrifying  bacteria  will  grow  vigorously  with  citrates  as  the  only 
source  of  organic  nutrients.  The  fermentation  of  lactates  by  butyric 
bacteria  has  been  known  for  a  long  time.  The  decomposition  of 
malates,  succinates,  tartrates  and  valerates  may  be  accomplished  by 
various  species,  and  even  simple  compounds  like  formates  may  yield 
food  and  energy  to  certain  soil  bacteria,  among  them  B.  methylicus 
studied  by  Loew  and  his  associates.  It  is  evident,  therefore,  that 
organic  acids  are  not  liable  to  accumulate  in  well- ventilated  soils. 
Molds,  as  well  as  bacteria,  destroy  them  rapidly,  and  carbonates, 
carbon  dioxide  and  water  are  the  final  products  of  the  decomposition 
of  non-nitrogenous  organic  matter. 

Notwithstanding  the  ready  decomposition  of  the  more  simple 
organic  acids  in  the  soil,  it  is  well  known  that  arable  soils  are  frequently 
acid.  This  acidity  is  largely  due  to  the  so-called  "humic  acids," 
organic  compounds  whose  composition  is  not  well  understood.  They 
are  composed,  to  some  extent,  of  rather  complex  organic  acids  or  of  their 
acid  salts.  Continued  cultivation  seems  to  favor  the  accumulation  of 
these  acid  compounds,  partly  on  account  of  the  diminished  supply  of 
lime  and  of  other  basic  materials  in  older  soils.  When  these  soils  are 
limed  the  humic  acids  and  acid  humates  are  changed  into  neutral  com- 
pounds and  are  then  subject  to  more  rapid  decomposition  by  micro- 
organisms. According  to  the  investigations  of  Blair  the  average  acid 
soil  in  Florida  requires  1,500  pounds  of  lime  (CaO)  per  acre  to  neutralize 
the  acidity  to  a  depth  of  84  mm.  (9  inches),  This  means  an  acidity 
equivalent  to  more  than  one  ton  of  hydrochloric  acid  per  acre.  In 
peat  and  muck  soils  the  acidity  is  equivalent  to  many  times  this 
amount  of  hydrochloric  acid. 

PROTEIN  BODIES  4 

Amount  and  Quality. — The  protein  content  of  farm  crops  that 
leave  residues  in  the  soil  is  variable,  but  in  all  cases  quite  considerable. 
Dried  corn  stalks  contain  5  per  cent  of  protein,  timothy  hay  6  per  cent, 
red  clover  hay  12  per  cent  or  more,  alfalfa  hay  15  or  16  per  cent.  Even 
wheat  and  rye  straw  may  contain  as  much  as  3  per  cent  of  protein. 
Cotton-seed  meal  and  other  oil  cakes,  tankage,  ground  fish,  hair  and 


DECOMPOSITION   OF   ORGANIC  MATTER   IN  THE   SOIL         381 

wool  waste  and  dried  blood  (all  used  more  or  less  extensively  as  sources 
of  nitrogen  to  crops)  are  made  up  in  a  large  measure  of  protein 
compounds. 

Being  derived  from  plant  residues,  from  microorganic,  insect  and 
animal  remains,  and  from  fertilizers  and  manures  applied,  the  nitrogen 
in  the  soil  humus  exists,  for  the  most  part,  in  the  form  of  protein  com- 
pounds. Hilgard  reports  the  following  humus  and  nitrogen  content, 
as  based  on  the  analyses  of  a  large  number  of  samples  of  humid, 
semi-arid  and  arid  soils. 


(Humus), 
per  cent 

(Nitrogen  in 
humus), 
per  cent 

(Nitrogen  in 
soil), 
per  cent 

Arid  uplands  ... 

O   01 

I">    23 

O   isc 

Sub-irrigated  arid  soils  

1.  06 

8.38 

O.OQQ 

Humid   soils   from   humid   and   arid   regions 
(California)  

2.4.1; 

C.  2O 

0.  11? 

Humid  soils  from  other  states     

7   OI 

3    ?8 

O.  2O^ 

Taking  the  weight  of  an  acre-foot  of  dry  soil  at  2,000,000  kg. 
(4,000,000  pounds)  and  multiplying  the  nitrogen  by  6.25  (the  factor 
usually  employed  for  converting  nitrogen  into  protein)  we  find  the 
protein  content  of  these  soils  to  range  from  about  11,339  kg.  (25,000 
pounds)  per  acre  to  nearly  three  times  as  much.  Similarly,  the 
Illinois  Experiment  Station  reports  quantities  of  nitrogen  equivalent 
to  3^73  to  4,989  kg.  (7,000  to  11,000  pounds)  per  acre  to  a  depth  of 
1 01. 6  cm.  (40  inches)  in  gray  silt  loams,  of  the  lower  Illinoisan  glacia- 
tion.  In  the  brown  silt  loams  the  amount  of  nitrogen  to  the  same  depth 
is  usually  more  than  4,535  kg.  (10,000  pounds)  per  acre;  occasionally 
it  is  more  than  9,071  kg.  (20,000  pounds)  per  acre.  In  one  instance  a 
black  clay  loam  of  the  late  Wisconsin  glaciation  is  reported  to  have 
about  13, 1 54  kg.  (29,000  pounds)  of  nitrogen  per  acre,  to  a  depth  of 
101.6  cm.  (40  inches).  This  would  be  equivalent  to  more  than  81,646 
kg.  (180,000  pounds)  of  protein;  of  course,  not  all  of  the  nitrogen  in  the 
soil  exists  in  the  form  of  protein,  some  of  it  occurring  as  amino-com- 
pounds,  and  a  small  portion  as  ammonia  and  nitrates.  Nevertheless, 
by  far  the  greatest  part  of  it  occurs  as  protein  compounds. 

The  protein  compounds  of  the  soil  humus  must  be  considered  from 
the  standpoint  of  quality  as  well  as  from  the  standpoint  of  quantity. 
It  is  well  known  that  fresh  plant  residues  are  attacked  more  readily  by 


382  MICROBIOLOGY   OF   SOIL 

microorganisms  than  older  plant  substances.  For  this  reason  soils 
frequently  supplied  with  fresh  organic  material  supply  greater  amounts 
of  available  food  to  crops  than  similar  soils  whose  organic  matter  con- 
sists largely  of  older  residues. 

Carbon-nitrogen  Ratio. — The  decomposition  of  organic  matter  is 
readily  influenced  by  the  relative  content  of  nitrogenous  and  non-ni- 
trogenous compounds.  Substances  of  animal  origin  yield  relatively  and 
absolutely  more  available  nitrogen  in  a  given  length  of  time  than  sub- 
stances of  plant  origin.  The  difference  noted  is  due  largely  to  the 
greater  proportion  of  protein  in  the  animal  materials;  in  other  words, 
to  the  narrower  carbon-nitrogen  ratio.  On  this  basis  Hilgard  attempts 
to  explain  the  adequacy  of  the  small  proportion  of  humus  in  arid 
and  semi-arid  soils.  Because  of  the  narrower  carbon-nitrogen  ratio 
the  humus  compounds  in  these  soils  are  decomposed  with  greater 
rapidity  and  yield  a  sufficient  amount  of  ammonia  and  nitrate  to  supply 
the  needs  of  the  crop. 

But  when  plant  substances  alone  are  considered  the  statement  just 
made  requires  qualification.  It  is  true  that  cotton-seed  meal  or  linseed 
meal,  having  a  narrower  carbon-nitrogen  ratio,  will  decay  more  readily 
than  corn-meal  or  wheat  flour.  It  is  also  true  that  any  given  plant  sub- 
stance, as  it  undergoes  decay,  will  lose  in  proportion  more  carbon  than 
nitrogen.  Older  humus  has,  therefore,  a  narrower  carbon-nitrogen 
ratio  than  humus  of  recent  origin.  The  former  is  more  resistant  to 
decay,  however,  than  new  humus.  In  a  concrete  way,  on  the  other 
hand,  it  may  be  stated  that  fresh  vegetable  material  of  a  narrow  car- 
bon-nitrogen ratio  will  decay  more  rapidly  than  fresh  vegetable  material 
of  a  wide  carbon-nitrogen  ratio.  The  reverse,  nevertheless,  is  true  of 
vegetable  materials  in  advanced  stages  of  decay.  Under  any  given 
climatic  conditions  and  in  any  given  soil  type,  the  carbon-nitrogen 
ratio  may  give  important  indications  only  as  to  the  availability  of  the 
humus  nitrogen.  Lawes  and  Gilbert,  as  quoted  by  Hall,  found  the 
following  carbon-nitrogen  ratio  in  the  organic  matter  of  different  soils : 

Cereal  roots  and  stubble 43  .o 

Leguminous  stubble 23 .  o 

Dung 18.0 

Very  old  grass  land *3  •  7 

Manitoba  prairie  soils 13 .o 

Pasture  recently  laid  down n-7 

Arable  soil 10.  i 

Clay  subsoil.. 6.0 


DECOMPOSITION    OF    ORGANIC   MATTER    IN    THE    SOIL          383 

Hall  concludes,  therefore,  that  humus  with  a  wide  carbon-nitrogen 
ratio  is  more  valuable  than  humus  with  a  narrow  carbon-nitrogen  ratio, 
since  the  latter  will  be  attacked  more  easily  by  the  soil  bacteria.  Brown 
and  Allison  indicate  that  there  might  be  a  possibility  of  applying  ma- 
terials of  a  wide  carbon-nitrogen  ratio  to  supply  the  deficiencies  of 
organic  matter  on  the  basis  that  the  former  may  have  the  same  or 
better  effect  on  bacterial  activities  such  as  azofication,  or  non-symbiotic 
nitrogen  fixation. 

THE  TRANSFORMATION  OF  NITROGEN  COMPOUNDS 
AMMONIFICATION.  Experimental  Study. — By  ammonification  is 
meant  the  production  of  ammonia  by  bacteria  out  of  protein  substances 
or  their  cleavage  products.  That  ammonia  production  in  the  soil  is 
a  biological  process  was  first  demonstrated  by  Miintz  and  Coudon  in 
1893.  These  investigators  showed  that  no  ammonia  is  formed  in  sterile 
soils.  They  also  showed  that  ammonia  may  be  produced  out  of  nitro- 
genous organic  matter  by  molds  as  well  as  by  bacteria.  Marchal  not 
only  confirmed  these  observations,  but  proved  that  various  micro- 
organisms differ  markedly  in  their  ability  to  produce  ammonia.  Of 
the  several  species  of  bacteria  tested  by  him,  B.  mycoides  (one  of  the 
common  soil  bacteria)  proved  itself  particularly  efficient  in  the  breaking 
down  of  nitrogenous  materials  and  the  production  of  ammonia. 

Since  the  publication  of  these  experiments  a  large  number  of  investi- 
gators, both  in  Europe  and  America,  have  studied  ammonia  production 
in  culture  solutions  as  well  as  in  the  soil  itself.  It  has  been  shown  that 
under  favorable  conditions  the  breaking  down  of  protein  compounds  and 
the  formation  of  ammonia  may  be  very  rapid;  for  instance,  in  some  ex- 
periments carried  out  by  Lipman  and  his  associates  the  following  pro- 
portions of  nitrogen  were  transformed  into  ammonia  in  the  course  of 
six  days: 

Dried  blood 16 . 74  per  cent 

Concentrated  tankage 56.66  per  cent 

Ground  fish 47 . 16  per  cent 

Cotton-seed  meal 4-95  per  cent 

Bone  meal 16 . 65  per  cent 

Cow  manure,  solid  and  liquid  excreta 32.60  per  cent 

Cow  manure,  solid  excreta 5-39  per  cent 

The  experiments  were  carried  out  in  equal  quantities  of  soil  and  with 
equivalent  quantities  of  nitrogen  in  the  different  substances.  It  will 


384  MICROBIOLOGY    OF    SOIL 

be  observed  that  more  than  56  per  cent  of  the  nitrogen  in  the  con- 
centrated tankage  was  transformed  into  ammonia,  whereas  under  the 
same  conditions  cotton-seed  meal  yielded  less  than  5  per  cent. 

Mechanism  of  Ammonia  Production. — The  relatively  large  protein 
molecules  are  readily  broken  into  larger  or  smaller  fragments.  This 
may  be  accomplished  by  purely  chemical  means,  as,  for  instance,  by 
boiling  with  acids  or  alkalies,  or  by  biological  activities.  Among  the 
first  cleavage  products  albumoses  and  peptones  are  quite  prominent. 
These  in  turn  undergo  further  cleavage  and  the  various  amino-acids 
and  their  derivatives,  as  well  as  ammonia,  make  their  appearance.  In 
so  far  as  the  different  species  of  bacteria  are  concerned,  the  hydrolysis  of 
proteins  seems  to  depend,  to  a  marked  extent,  on  the  ability  to  secrete 
proteolytic  enzymes.  With  the  aid  of  such  enzymes  the  proteins  are 
more  readily  hydrolyzed  and  further  changed  into  amino-  and  hydroxy- 
acids,  ammonia  and  carbon  dioxide. 

Influence  of  Soil  and  Climatic  Conditions. — Ammonia  production  in 
the  soil  is  affected  by  (a)  its  mechanical  and  chemical  composition;  by 
(b)  the  amount  and  distribution  of  rainfall;  by  (c)  the  prevailing  tem- 
peratures; by  (d)  fertilizer  treatment;  and  by  (e)  methods  of  tillage  and 
cropping.  The  mechanical  composition  of  the  soil  influences  the  pro- 
portion of  aerobic  and  anaerobic  species,  while  the  chemical  composi- 
tion, particularly  that  of  the  humus,  influences  the  rate  of  multiplica- 
tion and  the  character  of  the  chemical  transformation  accomplished. 
It  is  well  known,  for  example,  that  additions  of  fresh  organic  matter 
intensify  the  rate  of  decomposition  of  the  soil  humus,  and,  likewise, 
ammonia  production  as  has  been  already  demonstrated  by  Breal.  In  a 
more  general  way  it  was  proved  by  Lipman  and  his  associates  that, 
with  a  constant  bacterial  factor,  ammonia  production  in  soils  varies  with 
the  chemical  and  mechanical  composition  of  the  latter.  In  some  of 
these  experiments  100  g.  portions  of  different  soils  were  each  mixed  with 
5  g.  of  dried  blood,  sterilized  in  the  autoclave,  cooled  and  inoculated 
with  equal  quantities  of  infusion  from  fresh  soil.  The  following 
amounts  of  ammonia  nitrogen  were  produced  in  six  days: 

Soil  Ammonia  nitrogen  found 

A 31-62  mg. 

B . .     68. 29  mg. 

C 117.06  mg. 

D ; 107.16  mg. 

E.. 156.47  nig. 


DECOMPOSITION    OF    ORGANIC    MATTER   IN    THE    SOIL          385 

With  all  other  factors  constant,  chemical  and  mechanical  differences 
in  the  soil  used  were  responsible  for  striking  variations  in  ammonia 
production,  as  indicated  by  the  figures  given  above. 

The  influence  of  temperature  and  moisture  conditions  is  fully  as 
important  as  that  of  the  chemical  and  mechanical  composition  of  the 
soil.  The  following  data  secured  by  Lipman  may  be  cited  in  this 
connection  as  showing  the  effect  of  moisture: 

One-hundred-gram  quantities  of  air-dried  soil  were  each  mixed  with 
3  g.  of  dried  blood  and  varying  amounts  of  water  added.  The  ammonia 
formed  was  distilled  off  and  determined  at  the  end  of  eight  days. 
The  amounts  of  ammonia  nitrogen  found  were  as  follows: 

Water  added  Ammonia  nitrogen  found 

0  c.c .      4.13  mg. 

1  c.c. 4.13  mg. 

3  c.c 5.40  mg. 

5  c.c 10.64  mg. 

7  c.c 26 . 37  mg. 

10  c.c 49-57  mg. 

12  c.c 70.71  mg. 

IS  c.c 93.90  mg. 

It  appears,  therefore,  that  ammonia  production  in  soils  rises  or  falls 
as  the  rainfall  or  irrigation  is  increased  or  decreased,  or  as  the  soil  water 
is  more  or  less  thoroughly  conserved  by  proper  methods  of  tillage.  In 
the  same  way,  seasons  of  high  temperature  favor  ammonification  while 
seasons  of  low  temperatures  discourage  it.  This  point  is  well  illustrated 
by  the  observations  of  Marchal  that  at  o°  to  5°  only  traces  of  ammonia 
were  formed  in  his  culture  solutions;  that  at  20°  ammonia  production 
was  quite  marked,  and  that  at  30°  the  maximum  was  reached.  More- 
over, apart  from  the  seasonal  variations  in  any  one  locality,  there  is  a 
wide  range  in  ammonia  production,  as  we  pass  from  the  torrid  to  the 
temperate  and  from  the  latter  to  the  frigid  zones. 

Species  and  Numbers. — Ammonia  production  is  a  function  common 
to  most  soil  bacteria.  In  the  earlier  experiments  of  Marchal,  seventeen 
out  of  the  thirty-one  species  tested  were  found  capable  of  producing 
ammonia.  Prominent  among  these  ammonifiers  were  B.  mycoides, 
B.  (Proteus)  vulgaris,  B.  mesentericus  vulgatus,  B.  janthinus,  and 
B.  subtilis.  Of  a  considerable  number  of  soil  bacteria  tested  by 
Chester  all  but  one  were  observed  to  produce  ammonia.  In  Gage's 
experiments  with  sewage  bacteria,  seventeen  out  of  twenty  species 

25 


386  MICROBIOLOGY   OF   SOIL 

tested  proved  to  be  ammonifiers.  Similarly,  a  number  of  species  tested 
by  the  writer,  among  them  B.  coli,  B.  cholera  suis,  B.  (Proteus)  vulgaris, 
B.  subtilis,  B.  megatherium,  etc.,  all  produced  ammonia  in  meat  infusions. 
A  mass  of  additional  data,  accumulated  by  different  investigators, 
furnishes  further  proof  that  ammonia  production  is  a  common  function 
of  soil  bacteria. 

The  more  prominent  ammonifiers,  including  members  of  the  B. 
subtilis  group  and  certain  strep tothrices,  are  numerically  important  in 
all  arable  soils.  Their  numbers  are  affected,  however,  by  the  amount 
and  composition  of  the  soil  humus.  It  has  been  found,  for  instance, 
that  additions  of  straw  and  of  strawy  manure  increase  markedly  the 
numbers  of  B.  subtilis  and  of  other  members  of  the  group.  An  increase 
in  the  numbers  of  certain  ammonifiers  is  caused  also  by  additions  of 
lime  or  of  green  manure.  For  example,  in  experiments  carried  out  by 
Lipman  and  his  associates  portions  of  fertile  soil  inoculated  with  B. 
mycoides  were  found  to  contain,  a  month  later,  2,000,000  of  bacteria  per 
g.  of  soil.  In  similar  soil  portions  that  had  also  received  additions 
of  grass  the  number  was  twice  as  great. 

More  recent  investigations  (Temple,  Waksman)  have  shown  that 
ammonification  tests  are  of  little  value  in  determining  the  nature  of  the 
microbial  soil  flora,  since  the  rate  of  ammonia  production  is  largely 
controlled  by  the  soil  medium.  If  the  soil  is  suitable,  there  will  usually 
be  found  enough  microorganisms  capable  of  changing  the  protein  nitro- 
gen into  ammonia.  Temple  has  suggested  the  use  of  ammonification 
as  a  test  for  soil  fitness. 

Ammonification  should  be  studied  not  only  in  the  light  of  decompo- 
sition proteins  and  protein  derivatives  in  the  soil,  but  also  from  the 
point  of  view  of  energy  sources  in  the  soil.  Microorganisms  can  use 
both  carbohydrates  and  proteins  as  sources  of  energy.  There  is  a  great 
deal  more  of  the  carbon  compounds  oxidized  to  supply  the  required 
energy  than  there  is  nitrogen  consumed  in  the  normal  metabolism  of 
the  microbe.  The  addition  to  any  soil  of  definite  amounts  of  protein 
with  varying  amounts  of  available  carbohydrates  will  lead  to  the 
following  results :  ammonia  will  be  accumulated  in  the  soil  to  which  the 
protein  alone  has  been  added,  the  amounts  of  ammonia  increasing  with 
the  period  of  incubation  up  to  a  certain  point;  where  only  small  quanti- 
ties of  carbohydrates  have  been  added  there  will  be  at  first  no  ammonia 
produced,  but  soon  the  ammonia  will  begin  to  accumulate,,  so  that  the 
actual  quantities  of  ammonia  may  become  in  a  few  days  even  greater 


DECOMPOSITION   OF   ORGANIC   MATTER   IN   THE   SOIL          387 

than  in  the  soil  where  no  carbohydrates  have  been  added;  in  soils  to 
which,  aside  from  the  protein,  large  amounts  of  available  carbohydrates 
have  been  added,  no  ammonia  or  only  traces  of  it  will  be  found. 

Ammonia  is  produced  by  microorganisms  chiefly  in  the  deanniza- 
tion  of  the  amino  acids;  when  the  carbon  part  of  the  molecule  is  used  to 
supply  the  energy  required  and  the  nitrogen  part  is  not  consumed  in  the 
process  of  metabolism,  it  is  left  as  a  waste  product  in  the  form  of 
ammonia.  When  there  is  in  the  soil,  in  addition  to  the  proteins  and 
protein  decomposition  products,  a  sufficient  amount  of  available  carbo- 
hydrates, the  microorganisms  will  use  the  latter  as  a  source  of  energy 
and  will  attack  the  proteins  only  in  so  far  as  they  need  nitrogen  for  their 
metabolism.  In  that  case  no  ammonia  will  accumulate  in  the  soil; 
such  as  is  produced  will  probably  be  assimilated  by  the  microbes. 
But,  when  there  is  an  insufficient  amount  of  available  carbohydrates, 
the  microorganisms  are  compelled  to  use  the  proteins  not  only  as  sources 
of  nitrogen,  but  also  as  sources  of  energy.  More  carbon  will  then  be 
oxidized  to  supply  the  necessary  energy  than  there  will  be  nitrogen 
consumed;  the  excess  of  nitrogen  will  be  left  in  the  medium  as  a  waste 
product  in  the  form  of  ammonia.  The  presence  of  only  small  amounts 
of  available  carbohydrates  will  check  for  a  short  period  the  accumula- 
tion of  ammonia,  but  will  also  result  in  more  active  microbial  flora 
The  latter,  after  all  the  carbohydrate  is  used  up,  will  attack  the  proteins 
present  and  may  produce  larger  quantities  of  ammonia  than  if  no 
carbohydrate  had  been  added. 

Rate  of  Ammonia  Production. — Miyake,  using  the  results  obtained 
by  Lipman,  and  Waksman,  in  his  work  on  the  ammonia  production 
by  Aspergillus  niger,  have  shown  that  the  rate  of  ammonia  accumulation, 
whether  by  a  pure  culture  or  by  a  mixed  culture,  is  an  autocatalytic 
reaction.  The  rate  of  ammonia  accumulation  is  at  first  slow,  then  it 
begins  to  fall  off  and  finally  comes  to  a  standstill. 

Relative  Efficiency  of  Different  Species. — In  Marchal's  experiments 
already  referred  to,  the  species  employed  showed  marked  differences  in 
their  ability  to  produce  ammonia  out  of  egg  albumin.  The  following 
proportions  of  the  protein  nitrogen  were  converted  into  ammonia  in 
twenty  days: 

B.  mycoides 46  per  cent          B.  subtilis 23  per  cent 

B.  (Proteus)  vulgaris 36  per  cent          B.janthinus 23  per  cent 

B.  mesentericus  vulgatus..  29  per  cent          B.  fluorescens  putidus 22  per  cent 

Sarcina  lutea 27  per  cent  B .  fluorescens  liquefaciens  .  16  per  cent 


MICROBIOLOGY    OF    SOIL 

Furthermore,  apart  from  the  variations  from  species  to  species,  differ- 
ences have  been  observed  by  Marchal  and  many  other  investigators 
between  one  strain  and  another  of  any  single  species  isolated  from  the 
same  or  different  soils.  It  must  be  remembered,  therefore,  that  in  the 
study  of  ammonification  in  soils  and  culture  solutions,  due  considera- 
tion should  be  given  to  differences  in  physiological  efficiency  as  they  are 
manifested  by  strains  and  species  of  microorganisms. 

Apart  from  the  ammonifying  bacteria  already  mentioned  there  is  a 
group  of  organisms  studied  by  Muller,  Pasteur,  van  Tieghem,  Leube, 
Miquel,  Beyerinck  and  others.  These  are  the  so-called  urea  bacteria, 
capable  of  intensive  transformation  of  urea  and  allied  compounds  into 
ammonium  carbonate,  by  means  of  the  enzyme  urease. 

NH2 

CO  +  2H2O  =  (NH4)2CO3 

NH2 

Morphologicall  ythese  organisms  include  spherical  and  rod  forms, 
spore-bearing  and  non-spore  bearing  species.  Most  of  the  urea  bacteria 
are  particularly  prominent  in  the  transformation  of  animal  manures. 

Ammonifying  Efficiency. — Lipman  and  Burgess  have  found  marked 
differences  in  the  ammonifying  efficiency  of  fifteen  organisms  in  pure 
cultures  using  peptone,  bat  guano,  sheep  and  goat  manure,  dried 
blood,  tankage,  cottonseed  meal  and  fish  guano.  The  nature  of  the 
soil  as  well  as  the  nature  of  the  nitrogenous  material  markedly  modify 
an  organism's  ammonifying  power.  B.  tumescens  on  the  whole  appears  to 
have  been  the  most  efficient  organism  tested.  Comparing  these  findings 
with  those  of  Marchal  the  former  have  obtained  results  in  soils,  while 
the  latter 's  work  was  with  solution  cultures,  the  application  of  which 
to  soil  conditions  is  not  always  permissible.  In  point  of  fact  the  am- 
monifying efficiency  of  organisms  is  greater  in  sandy  soil  and  possi- 
bly in  others  than  in  solutions,  as  Lipman  and  Burgess  have  obtained 
a  transformation  of  41.98  per  cent  of  peptone  in  nitrogen  and  36.06 
per  cent  of  bat  guano  nitrogen  into  ammonia  by  Sarcina  lutea  and 
B.  mycoides,  respectively,  in  twelve  days  at  temperatures  between 
27°  and  30°,  while  Marchal  obtained  similar  transformations  in 
thirty  days  at  30°  in  albumen  solutions. 


DECOMPOSITION    OF    ORGANIC   MATTER   IN   THE    SOIL          389 

It  is  also  of  interest  to  note  that  investigations  with  soil  fungi  have 
revealed  the  fact  that  certain  species  are  even  more  efficient  am- 
monifiers  than  B.  mycoides.  McLean  and  Wilson,  Waksman,  Cole- 
man  and  Kopeloff  have  worked  with  organisms  like  Trichoderma 
koeningi  which  is  capable  of  transforming  more  than  50  per  cent  of 
the  nitrogenous  material  added  in  such  experimentation. 

NITRIFICATION.  Experimental  Study. — The  term  nitrification  refers 
to  the  oxidation  either  of  ammonia  or  of  nitrites  to  nitrates.  In  a 
broader  sense  nitrification  may  be  defined  as  the  production  of  nitrates 
from  decomposing  organic  matter.  Saltpeter  or  niter,  the  terms 
formerly  applied  to  potassium  nitrate,  possessed,  for  a  long  time,  a 
peculiar  interest  because  of  its  relation  to  gunpowder.  Whether  it  be 
true  or  not  that  gunpowder  was  known  to  the  Chinese  before  the  be- 
ginning of  the  present  era,  there  is  no  doubt  that  for  several  centuries 
it  played  an  important  part  in  the  political  and  economic  history  of 
Europe.  The  large  quantities  of  gunpowder  consumed  in  the  almost 
incessant  wars  created  a  steady  demand  for  saltpeter  that  was  not 
readily  met  by  the  saltpeter  refiners  of  India,  Hungary  and  Poland. 
European  nations,  particularly  France,  were  therefore  thrown  on  their 
own  resources  and  were  forced  to  develop  the  domestic  production  of 
saltpeter.  The  industry  came  under  government  control  and  experts 
were  appointed  to  study  the  so-called  saltpeter  plantations  and  the 
conditions  affecting  the  appearance  and  increase  of  nitrates  in  com- 
post heaps  and  in  the  soil.  Much  knowledge  was  thus  gained  about 
nitrification  even  though  it  was  not  suspected  that  living  organisms 
were  concerned  in  the  process. 

With  the  rapid  development  of  chemistry  in  the  latter  half  of  the 
eighteenth  century  a  nearer  approach  was  made  to  the  understanding 
of  the  true  character  of  nitrification.  The  observations  of  Cavendish 
in  1784  that  potassium  nitrate  is  formed  when  electric  sparks  are  passed 
through  air  confined  over  a  solution  of  potassium  hydrate  formed  the 
starting  point  for  various  theories  that  attempted  to  account  for  nitrate 
formation  on  the  basis  of  purely  chemical  reactions.  The  formation  of 
nitric  acid  and  of  its  salts  was  thus  assumed  to  be  due  to  electric  dis- 
charges in  the  atmosphere,  to  combustion  processes  in  nature,  or  to  the 
oxidation  of  organic  matter  and  of  calcium,  magnesium,  iron  and  man- 
ganese compounds  in  the  soil.  Much  credence  was  given  to  the  latter 
explanation  because  of  the  almost  universal  occurrence  of  nitrates  in 
arable  soils. 


MICROBIOLOGY   OF    SOIL 

The  first  indication  that  nitrate  production  in  the  soil  and  in  de- 
caying organic  matter  is  due  to  biological  activities  was  given  by 
Pasteur  in  1862.  A  few  years  later  M tiller  expressed  his  belief  in  the 
biological  origin  of  nitrates  and  nitrites  in  sewage  and  drinking  water. 
It  was  not,  however,  until  1877  that  the  true  character  of  nitrification 
was  made  clear.  In  that  year  Schloesing  and  Miintz  demonstrated 
that  dilute  solutions  of  ammonia  could  be  changed  into  nitrate  by  being 
passed  slowly  through  long  tubes  filled  with  soil.  The  amounts  of 
nitrate  nitrogen  found  in  the  leachings  corresponded  almost  exactly 
to  the  amount  of  ammonia  nitrogen  used  up.  When  the  soil  in  the 
tubes  was  first  sterilized  by  heating  or  by  means  of  chloroform  and  other 
germicides,  the  ammonia  passed  through  unchanged.  But  when  soils 
sterilized  by  heat  or  chloroform  were  reinfected  with  small  quantities 
of  fresh  soils  nitrification  again  proceeded  in  a  normal  manner. 

The  biological  nature  of  nitrification  having  been  thus  established 
numerous  investigators  tried  to  isolate  the  specific  organisms  in  pure 
culture.  A  large,  amount  of  work  in  this  direction  was  done  by 
Schloesing  and  Miintz,  Celli  and  Marino-Zuco,  Munro,  Warington,  the 
Franklands  and  many  others.  A  large  number  of  bacteria,  yeasts  and 
molds  were  tested  with  negative  results.  Warington,  who  gathered 
a  great  mass  of  valuable  information  about  nitrification,  almost 
succeeded  in  securing  pure  cultures  of  nitrifying  bacteria.  Finally, 
Winogradski  showed  in  1890  not  only  that  nitrification  is  caused  by 
specific  bacteria,  but  explained  also  why  the  others  failed  in  securing 
pure  cultures.  He  proved  that  these  organisms  do  not  develop  colonies 
on  the  ordinary  gelatin  and  other  organic  media,  a  fact  whose  recog- 
nition was  largely  responsible  for  his  successful  solution  of  the  problem. 
The  medium  subsequently  employed  by  him  consisted  of  silica  jelly 
properly  supplied  with  inorganic  nutrient  salts.  After  him  other  in- 
vestigators proved  that  agar,  deprived  of  its  soluble  organic  matter, 
gypsum  and  sandstone  disks,  filter-paper  pads,  etc.,  could  be  used 
effectively  as  solid  media. 

Nitrous  and  Nitric  Bacteria. — Winogradski' s  investigations  led  to 
the  conclusion,  foreshadowed  by  the  earlier  work  of  the  Franklands  and 
Warington,  that  the  oxidation  of  ammonia  proceeds  in  two  stages,  viz., 

(1)  2NH3  +  302   =  2HN02  +  2H2O 

(2)  2HNO2  +  O2  =  2HNO3 


DECOMPOSITION    OF    ORGANIC   MATTER    IN    THE    SOIL          39 1 

The  organisms  oxidizing  ammonia  to  nitrites,  and  designated  as 
nitrous  or  nitrite  bacteria,  were  called  by  Winogradski  Nitrosomonas 
and  Nitrosococcus.  The  former  include  species  or  varieties  isolated 
from  soils  in  Europe,  Asia  and  Africa,  and  the  latter  those  isolated  from 
soils  in  America  and  Australia.  The  organisms  oxidizing  nitrites  to 
nitrates  and  known  as  nitric  or  nitrate  bacteria,  were  included  by 
Winogradski  in  the  genus  Nitrobacter. 

Apart  from  these  bacteria  there  is  an  organism,  according  to  Kaserer, 
that  can  oxidize  ammonia  directly  to  nitrate.  He  named  it  B.  nitrator. 
The  reaction  is  illustrated  by  the  following  equation: 

NH3  +  H2CO3  +  O2  =  HNO3  +  H2O  +  CH2O  -  41  Cal. 
CH2O  +  O2  =  H2CO3  +  132  Cal. 

Enough  energy  for  the  completion  of  the  reaction  is  obtained  by  the 
oxidation  of  the  formaldehyde  (CH2O).  Beyond  tfie  preliminary 
announcement  of  Kaserer's  there  are  no  experimental  data  to  prove 
the  existence  of  this  organism,  even  though  other  evidence  of  an 
indirect  nature  may  be  construed  to  lend  support  to  his  theory. 
But  whether  it  be  proved  or  not  that  ammonia  may  be  oxidized 
to  nitrate  by  a  single  species,  it  is  evident  that  the  number  of  species 
concerned  in  nitrate  production  is  relatively  small. 

Relation  to  Environment. — The  conditions  that  affect  nitrate  forma- 
tion in  soils  may  be  classified  under  the  following  heads:  (a)  supply  of 
oxygen;  (b)  range  of  prevailing  temperatures;  (c)  amount  and  dis- 
tribution of  moisture;  (d)  quantity  of  lime  and  of  other  basic  materials; 
(e]  quantity  of  soluble  mineral  salts;  (/)  character  and  amount  of 
organic  matter;  (g)  presence  of  toxic  substances;  (h)  association  with 
other  organisms;  (i)  physiological  efficiency  of  the  nitrifying  bacteria. 

The  rapid  disappearance  of  organic  matter  from  sandy  soils  is  due  in 
large  measure  to  their  better  aeration.  On  the  other  hand,  the  decom- 
position of  vegetable  and  animal  substances  in  heavy,  ill-ventilated  soils 
is  materially  retarded  by  the  limited  supply  and  very  gradual  renewal  of 
oxygen.  An  intimate  relation  exists  here  between  air  and  water  in  that 
the  latter  replaces  the  former  to  a  more  marked  extent  in  heavy  than  in 
light  soils.  The  influence  of  both  aeration  and  the  range  of  moisture  is 
illustrated  by  an  experiment  of  Lipman's  in  which  equal  quantities  of 
soil  were  kept  in  large  boxes  under  different  moisture  conditions.  At 


392  MICROBIOLOGY   OF   SOIL 

the  end  of  a  year  the  following  quantities  of  nitrate  nitrogen  were 
found: 


Moisture 
content 

Nitrate 
nitrogen 
found 


6.52  per  cent  14.75  Per  cent  18.62  per  cent  22.05  Per  cent  22.12  per  cent 


697  mg.  823  mg.  720  mg.  Trace  Trace 


In  examining  the  figures  recorded  above,  we  find  that  moisture  was  the 
controlling  factor  in  the  development  of  the  nitrifying  bacteria,  when 
the  proportion  of  water  in  the  soil  was  6.52  per  cent.  As  the  amount  of 
water  increased  to  14.75  Per  cent  there  was  a  marked  increase  in  the 
amount  of  nitrate  produced.  Beyond  that,  however,  the  further  in- 
crease in  the  amount  of  water  began  to  limit  the  supply  of  oxygen,  and 
the  production  of  nitrate  nitrogen  with  18.62  per  cent  of  water  in  the 
soil  was  somewhat  decreased.  A  still  further  addition  of  water  up  to 
22.05  Per  cent  led,  practically,  to  saturation,  and  the  encouragement  of 
reduction  rather  than  oxidation  processes.  Hence,  no  nitrate  was  al- 
lowed to  accumulate  in  the  soil.  The  data  in  question  thus  help  to 
explain  why  care  was  taken,  on  saltpeter  plantations,  to  keep  the 
compost  heaps  moist,  yet  not  too  wet. 

The  influence  of  temperature  on  nitrate  formation  has  been  observed 
by  many  investigators.  Schloesing  and  Mlintz  recorded  that  at  5° 
nitrification  is  quite  feeble,  at  12°  marked  and  at  37°  at  its  best. 
Other  investigators  have  obtained  substantially  the  same  results,  except 
that  the  optimum  has  been  found  to  be  considerably  lower,  often  be- 
tween 25°  and  30°.  Under  field  conditions  nitrification  seems  to  take 
place  at  relatively  low  temperatures,  as  is  indicated  by  the  rapid 
oxidation  of  ammonium  salts  in  the  Rothamsted  experiments  in  Eng- 
land; and  the  rapid  decay  and  nitrification  of  clover  and  of  other 
legume  residues  in  the  experiments  at  the  New  Jersey  Experiment 
Station.  These  facts  have,  therefore,  an  important  bearing  on  the 
nitrogen  feeding  of  crops  in  tropical,  subtropical  and  temperate  zones. 

The  influence  of  lime  and  of  other  basic  substances  including  the 
carbonates  of  magnesium,  potassium  and  sodium,  and  of  the  oxides  of 
iron  is  of  far-reaching  importance  in  all  nitrification  processes.  It  is 
well  known  that  applications  of  magnesian  and  non-magnesian  lime, 
marl  or  wood  ashes  promote  nitrification  in  the  soil  and  in  compost 
heaps,  a  fact  that  was  well  recognized  by  the  ancient  niter  refiners.  The 


DECOMPOSITION    OF    ORGANIC  -MATTER    IN   THE    SOIL          393 

favorable  action  of  lime  is  readily  explained  by  its  ability  to  neutralize 
organic  and  mineral  acids  and  to  render,  thereby,  the  soil  reaction 
favorable  for  the  rapid  growth  of  ammonifying,  as  well  as  of  nitrifying 
bacteria.  Furthermore,  the  reserve  of  basic  material  serves  to  neutral- 
ize the  acid  formed  by  the  bacteria  and  prevents  thus  the  accumulation 
of  an  undue  amount  of  acidity. 

The  role  of  certain  mineral  salts  in  promoting  nitrification  is  quite 
significant.  Small  amounts  of  sodium  chloride  have  been  found  to  favor 
nitrification  in  the  experiments  of  Pichard  and  also  those  of  Lipman. 
The  former  showed  also  that  sulphates  not  only  promote  nitrification, 
but  that  different  sulphates  display  marked  variations  in  this  respect. 
In  the  same  manner  nitrate  formation  was  shown  to  be  favorably 
affected  by  phosphates  in  bone  meal,  Thomas  slag,  and  acid  phos- 
phates. Generally  speaking,  therefore,  nitrifying  bacteria  are  stimu- 
lated in  their  development  by  a  proper  supply  of  available  mineral 
nutrients. 

The  exact  relation  of  organic  matter  in  the  soil  to  the  activities  of 
nitrifying  bacteria  is  but  beginning  to  be  properly  understood.  Earlier 
observations  made  it  manifest  that  heavy  applications  of  animal 
manures,  or  of  green  manure  may  not  only  retard  nitrification,  but  may 
actually  cause  the  disappearance  of  a  part  or  of  all  of  the  nitrate  in  the 
soil.  Subsequent  experiments  by  Winogradski  and  by  Winogradski 
and  Omelianski  showed  that  in  pure  cultures  the  presence  of  even  slight 
amounts  of  soluble  organic  matter  may  depress  or  even  suppress  the 
development  of  the  nitrifying  bacteria.  It  was,  therefore,  concluded 
by  these  authors  that  relatively  small  amounts  of  soluble  organic 
matter  may  inhibit  nitrification.  These  conclusions,  based  on  the 
study  of  liquid  cultures  only,  were  given  a  very  broad  application  by 
many  writers  on  agricultural  topics.  More  recent  experiments  make 
it  certain,  however,  that  in  the  soil  itself  small  amounts  of  soluble 
organic  matter,  e.g.,  dextrose,  are  not  only  harmless,  but  may  really 
stimulate  nitrification.  It  was  shown,  likewise,  that  humus  and 
extracts  of  humus  may,  under  suitable  conditions,  stimulate  nitrifica- 
tion to  a  very  striking  extent. 

Certain  substances  in  the  soil  may  exert  a  toxic  effect  on  nitrifying 
bacteria.  Ferrous  sulphate,  sulphites  and  sulphides  may  thus  act  in- 
juriously, as  may  also  calcium  chloride  and  excessive  concentrations  of 
sodium  carbonate,  sodium  bicarbonate,  sodium  chloride,  magnesium 


394  MICROBIOLOGY   OF   SOIL 

sulphate,  etc.  Injury  by  ferrous  compounds,  as  well  as  by  organic 
acids,  is  not  uncommon  in  low-lying  fields  and  bogs;  while  injury  from 
excessive  concentration  of  soluble  salts  may  occur  in  the  so-called 
alkali  lands. 

Finally  nitrification  in  the  soil  should  be  considered  from  the  stand- 
point of  the  organisms  themselves.  There  is  no  doubt  that  continued 
growth  under  extremely  favorable  conditions  leads  to  the  develop- 
ment in  the  soil  of  nitrifying  bacteria,  possessing  a  very  marked  phy- 
siological efficiency.  On  the  other  hand,  in  ill-aerated,  sour  soils  the 
environment  would  depress  the  physiological  efficiency  of  the  nitrify- 
ing bacteria.  Differences  are  thus  undoubtedly  established  under 
actual  field  conditions,  as  is  made  probable  by  the  variable  behavior 
of  soils  from  different  sources  when  used  as  inoculating  material  in 
recently  reclaimed  or  peat  swamp  lands. 

Accumulation  and  Disappearance  of  Nitrates. — As  shown  above,  the 
rate  of  formation  of  nitrates  in  the  soil  is  dependent  upon  moisture, 
temperature  and  aeration,  as  well  as  on  the  presence  of  organic  matter 
and  basic  substances.  On  the  other  hand,  the  accumulation  of  nitrates 
depends,  under  any  given  conditions,  largely  on  the  character  of  the 
growing  crop.  Observations  on  the  rain  gauges  at  Rothamsted  showed 
an  average  annual  loss  of  14  kg.  (31.4  pounds)  of  nitric  nitrogen  per  acre 
in  the  drainage  water  from  uncropped  soil.  In  one  of  King's  experi- 
ments, land  that  had  been  fallowed  contained  137  kg.  (303.24  pounds) 
of  nitric  nitrogen  per  acre,  to  a  depth  of  4  feet.  Adjoining  cropped 
land  contained  only  26  kg.  (57.56  pounds)  of  nitric  nitrogen  per  acre 
to  the  same  depth.  Stewart  and  Greaves  found  in  limestone  soil  in 
Utah  64  kg.  (142  pounds)  of  nitric  nitrogen  per  acre,  under  corn; 
98  pounds  under  potatoes,  and  only  12  kg.  (27  pounds)  under  alfalfa. 
Under  the  same  conditions  fallow  land  contained  74  kg.  (165  pounds) 
of  nitric  nitrogen  per  acre.  The  smaller  amount  of  nitric  nitrogen  found 
under  alfalfa  bears  out  the  observations  already  made  by  a  number  of 
other  investigators  that  the  accumulation  of  nitrates  under  legumes  is 
smaller  than  it  is  under  non-legumes.  While  several  explanations  have 
been  offered  to  account  for  this  fact,  it  is  generally  agreed  that  legumes 
assimilate  nitrate  nitrogen  more  rapidly  than  non-legumes.  Unusual 
circumstances  may  favor,  at  times,  the  accumulation  of  quantities  of 
nitrate  large  enough  to  destroy  all  vegetation.  It  is  reported,  for 


DECOMPOSITION   OF    ORGANIC    MATTER    IN    THE    SOIL          395 

instance,  by  Headden  that  he  has  found  in  limited  areas  in  Colorado  as 
much  as  90,718.5  kg.  (100  tons)  of  nitrate  per  acre  foot  of  soil. 

The  amount  of  nitrate  nitrogen  in  the  soil  is  influenced  by  the  grow- 
ing crop  not  alone  because  of  the  nitrogen  absorbed  by  the  latter,  but 
because  of  the  moisture  relations  as  affected  by  growing  plants.  It  is 
quite  apparent  that  a  large  crop  dries  out  the  soil  more  rapidly  than  a 
small  crop.  When  the  soil  moisture  is  sufficiently  depleted,  nitrifica- 
tion stops  and  the  further  accumulation  of  nitrates  becomes  impossible, 
while  their  disappearance  is  hastened  by  the  constant  demands  of  the 
crop.  The  disappearance  of  soil  nitrates  is,  likewise,  hastened  by  the 
leaching  action  of  rain  and  by  certain  species  of  bacteria  that  transform 
them  into  other  nitrogen  compounds. 

DENITRIFICATION.  Experimental  Study. — Denitrification  may  be 
defined  as  the  reduction  of  nitrates  by  bacteria,  involving  the  evolu- 
tion of  nitrogen  gas  or  of  nitrogen  oxides.  In  a  more  general  way, 
denitrification  has  been  defined  as  the  partial  or  complete  reduction  of 
nitrates  by  bacteria.  The  term  direct  denitrification  has  been  sug- 
gested for  complete  reduction,  and  indirect  for  the  partial  reduction 
to  nitrites  or  ammonia.  The  term  denitrification  should  not  be  em- 
ployed to  designate  losses  of  nitrogen  gas  due  to  the  oxidation  of 
ammonia,  or  to  the  disappearance  of  nitrates  following  their  conversion 
into  proteins  by  microorganisms. 

The  reduction  of  nitrates  in  the  presence  of  fermenting  organic 
matter  was  noted  by  Kuhlmann  as  early  as  1846.  The  same  fact  was 
recorded  many  years  later  by  Froehde  and  by  Angus  Smith.  In  1868 
Schoenbein  expressed  the -belief  that  nitrates  may  be  reduced  to  nitrites 
by  fungi.  For  more  than  a  decade  after  that,  data  were  rapidly  accu- 
mulating in  support  of  Schoenbein's  contention,  until  in  1882  Gayon 
and  Dupetit  made  it  certain  that  nitrate  reduction  with  the  evolution 
of  nitrogen  gas  may  be  caused  by  a  "ferment."  Finally,  in  1886,  the 
same  investigators  described  two  organisms,  B.  denitrificans  a,  and  B. 
denitrificans  0,  capable  of  completely  reducing  nitrates.  Subsequently 
the  studies  of  Giltay  and  Aberson,  Burri  and  Stutzer,  Severin,  van 
Iterson,  Jensen,  Beyerinck  and  of  many  others  not  only  greatly  in- 
creased the  number  of  known  denitrifying  bacteria,  but  added  much  to 
our  knowledge  concerning  the  development  and  activities  of  these 
organisms.  It  has  been  shown  that  a  very  large  number  of  species 
can  reduce  nitrates  to  nitrites  and  ammonia;  moreover,  a  considerable 


396  MICROBIOLOGY   OF   SOIL 

number  of  organisms  are  already  known  that  can  cause  the  complete 
destruction  of  nitrates  with  the  evolution  of  nitrogen  gas  or  nitrogen 
oxides.  The  following  reactions  illustrate  diagrammatically  the  com- 
plete or  partial  reduction  of  nitrates. 

2HNO3  =  2HNO2  +  O2 
HNO3  +  H2O  =  NH3  +  2O2 

4HNO2  =  2H2O  +  2N2  +  sO2 

In  the  soil,  manure  or  other  culture  media  the  denitrifying  bacteria 
which  are,  for  the  most  part,  aerobic  develop  also  under  anaerobic 
conditions  and  transfer  the  oxygen  of  nitrates  and  nitrites  to  carbon 
compounds.  This  is  illustrated  by  the  equations  suggested  by  van 
Iterson : 

5C  +  4KNO3  +  2H2O  =  4KH  CO3  +  2N2  +  CO2 
3C  +  4KNO2  +  H2O  =  2KH  CO3  +  K2CO3  +  2N2 

When  nitrates  are  reduced  to  nitrites  in  the  presence  of  amino- 
compounds,  or  even  of  ammonium  compounds,  elementary  nitrogen 
may  escape  as  shown  by  the  following  reactions: 

C2H5NH2  +  HNO2  =  C2H6OH  +  N2  +  H2O 
NH4C1  +  KNO2  =  KC1  +  2H20  +  N2 

An  organism  has  been  described  by  van  Iterson  that  can  decompose 
nitrates  in  the  presence  of  cellulose: 

5C6H10O5  +  24KNO3  =  24KHCO3  +  6CO2  +  i2N2  +  i3H2O 

Still  more  interesting  is  Thiobacillus  denitrificans  described  by 
Beyerinck  as  capable  of  reducing  nitrates  in  inorganic  media.  The 
nitrate  oxygen  is  used  to  oxidize  elementary  sulphur: 

6KNO3  +  5S  +  2CaCO3  =  3K2SO4  +  2CaSO4  +  2CO2  +  3N2 

The  Actinomyces  reduce  nitrates  to  nitrites,  but  they  do  not  cause 
any  loss  of  free  nitrogen,  for  the  nitrites  are  utilized  by  the  organisms. 
and  complete  denitrification  does  not  take  place.  Thus  these  organ- 
isms may  prevent  the  leaching  out  of  nitrates  and  nitrites  in  the  soil, 
or  the  active  denitrification  by  other  organisms. 

Relation  to  Environment. — Nitrate  reduction  is  favored  by  insuffi- 
cient aeration,  as  well  as  by  an  abundance  of  readily  decomposable 
organic  matter.  In  fine-grained,  compact  soils  nitrate  formation  and 
nitrate  reduction  may  alternate,  depending  upon  the  more  or  less 


DECOMPOSITION    OF    ORGANIC   MATTER    IN    THE    SOIL          397 

complete  replacement  of  soil  air  by  water.  Similarly,  in  soils  receiving 
excessive  amounts  of  animal  manure  denitrifying  bacteria  may  cause 
the  reduction  of  nitrates.  In  greenhouse  soils  excessive  moisture,  as 
well  as  excessive  amounts  of  organic  matter,  may  be  present  and  may 
prevent  the  accumulation  of  nitrates.  It  has  also  been  shown  by 
Niklevski  that,  contrary  to  opinions  previously  held,  denitrification 
may  occur  in  manure  heaps.  In  the  better  aerated  surface  portion  of 
manure  heaps  conditions  are  favorable  for  the  oxidation  of  ammonia 
to  nitrites  and  nitrates.  The  nitrous  acid  may  combine  with  ammonia 
to  form  ammonium  nitrite,  the  latter  decomposing,  spontaneously,  into 
water  and  nitrogen  gas.  It  is  very  likely,  also,  that  the  nitrites  and 
nitrates  are  reduced  by  the  denitrifying  bacteria  in  manure.  On  the 
other  hand,  in  manure  kept  moist  under  the  feet  of  cattle  nitrite  and 
nitrate  formation  is  prevented  and  losses  by  denitrification  are  not 
likely  to  occur. 

The  economic  significance  of  denitrification  was  overestimated  at 
one  time,  on  account,  largely,  of  the  assertion  of  Wagner  in  1895  that 
in  all  soils  receiving  applications  of  horse  manure,  the  nitrates  in  the 
soil  itself  as  well  as  those  added  in  commerical  fertilizers  are  almost 
certain  to  be  destroyed  by  denitrification.  Subsequent  experiments 
by  many  investigators  demonstrated  that  under  field  conditions,  deni- 
trification is  a  factor  of  slight  moment;  however,  in  the  greenhouse, 
in  the  manure  heap  (under  certain  conditions)  and  in  market  gardening 
where  manure  is  used  at  the  rate  of  45,359  kg.  to  54,431  kg.  (50  to  60 
tons)  per  acre,  the  danger  of  denitrification  is  real. 

ANALYTICAL  AND  SYNTHETICAL  REACTIONS 

AMOUNT  OF  BACTERIAL  SUBSTANCE  IN  THE  SOIL. — Various  decom- 
position processes  in  the  organic  matter  of  the  soil  may  be  designated 
as  analytical  in  that  protein,  carbohydrates  and  fats  are  split  into  more 
simple  compounds.  At  the  same  time,  the  microorganisms  concerned 
in  the  decomposition  processes  multiply  very  rapidly  and  fashion  the 
complex  compounds  of  their  cell-substance  out  of  the  simple  cleavage 
products  in  their  medium.  In  other  words,  analytical  and  synthetical 
reactions  proceed  hand  in  hand  in  the  soil. 

While  it  is  not  definitely  known  how  large  a  proportion  of  the  soil 
humus  consists  of  the  dead  and  living  cells  of  microorganisms  there 
is  a  mass  of  indirect  evidence  to  show  that  these  cells  form  a  very  con- 


MICROBIOLOGY   OF   SOIL 

siderable  proportion  of  the  total  quantity  of  organic  substances  in  the 
soil.  For  instance,  it  has  been  demonstrated  that  a  large  proportion  of 
the  dry  matter  of  solid  animal  faeces  may  consist  of  bacterial  cells.  At 
various  times  and  by  different  investigators  the  proportion  of  bacterial 
substance  has  been  estimated  at  from  5  to  20  per  cent  or  more  of  the 
total  dry  weight  of  faeces.  A  heavy  application  of  barnyard  manure 
may  introduce,  therefore,  several  hundred  pounds  of  bacterial  cells  per 
acre  of  soil.  Moreover,  because  of  the  extensive  changes  in  the  soil 
humus  itself,  as  is  evidenced  by  the  rapid  formation  of  nitrates,  large 
masses  of  bacterial  substances  are  constantly  being  formed  and  dis- 
integrated. 

AVAILABILITY  OF  BACTERIAL  MATTER. — Substances  of  microorganic 
origin  are  decomposed  more  or  less  rapidly,  according  to  their  com- 
position. The  extent  of  transformation  under  favorable  conditions  is 
indicated  by  an  experiment  performed  by  Beyerinck  and  van  Delden, 
in  which  50  per  cent  of  the  nitrogen  in  Azobacter  cells  was  transformed 
into  nitrate  in  seven  weeks.  On  the  other  hand,  the  humus  of  peat  and 
muck  soils,  or  that  of  worn-out  soils,  may  contain  microorganic  residues 
of  so  inert  a  character  as  to  yield  but  little  a  vailable_  nitrogen  to 
crops. 

TRANSFORMATION  OF  PEPTONE,  AMMONIA  AND  NITRATE  NITROGEN. 
The  cleavage  of  protein  compounds  into  peptones,  amino-acids  and 
ammonia,  and  the  oxidation  of  the  latter  into  nitrites  and  nitrates,  may 
be  properly  included  among  analytical  reactions.  It  should  not  be 
forgotten,  however,  that  in  the  accompanying  synthetical  reactions  the 
compounds  just  mentioned  may  be  transformed  back  into  complex 
proteins.  It  happens,  thus,  that  large  quantities  of  the  available 
nitrogen  compounds  may  be  withdrawn  from  circulation  by  micro- 
organisms that  use  these  as  building  material.  Under  extreme  con- 
ditions microorganisms  may  become  serious  competitors  of  higher 
plants  for  available  nitrogen  food. 

Manure  stored  in  heaps  not  infrequently  deteriorates  in  quality, 
even  when  losses  by  leaching  are  excluded.  This  deterioration  is  largely 
due  to  the  change  of  the  water-soluble  ammonia  and  amino-compounds 
into  insoluble  protein  substances.  While  the  extent  of  the  change  into 
protein  compounds  is  variable  it  may  range  from  less  than  a  tenth  of  the 
water  soluble  material  to  more  than  three-quarters  or  four-fifths  of  it. 
Also  in  the  soil  the  same  processes  take  place,  but  not  so  intensively.  A 


DECOMPOSITION    OF    ORGANIC   MATTER   IN   THE    SOIL         399 

large  number  of  species  of  molds  and  bacteria  have  been  isolated  and 
tested  as  to  their  ability  to  transform  ammonia,  amino-  and  nitrate 
nitrogen  into  protein  compounds.  Among  the  more  recent  investi- 
gations in  this  field  those  of  Lemmermann  and  his  associates  testify  that 
in  three  weeks  5  to  6  per  cent  of  the  nitrate  added  to  the  soil  was  changed 
into  protein.  In  the  presence  of  barnyard  manure  the  proportion 
transformed  was  increased  to  15  per  cent.  In  the  case  of  ammonium 
compounds  the  transformation  may  be  even  more  far-reaching,  amount- 
ing, at  times,  to  more  than  25  to  30  per  cent  of  the  material  originally 
present.  Generally  speaking,  molds  will  assimilate  ammonia  nitrogen 
more  readily  while  bacteria  and  algae  will  assimilate  nitrate  nitrogen 
by  preference.  However,  the  preference  of  molds  for  ammonia  nitrogen 
is  often  more  apparent  than  real,  because  of  the  rapid  formation  of 
acid  residues  in  culture  media  rich  in  certain  ammonium  compounds. 
Similarly,  some  species  of  bacteria  will  assimilate  ammonia  nitrogen 
in  preference  to  nitrate  nitrogen. 


CHAPTER  III 
FIXATION  OF  ATMOSPHERIC  NITROGEN 

THE  SOURCE  OF  NITROGEN  IN  SOILS 

EARLY  THEORIES. — When  chemistry  had  made  sufficient  progress 
to  allow  the  analysis  of  soils  and  plants  it  was  recognized  that  nitrogen 
is  always  present  in  both.  It  was  also  recognized  that  the  soil  nitrogen 
is  almost  wholly  confined  to  the  surface  portion  and  is  evidently  of 
atmospheric  origin,  since  the  unweathered,  underlying  rock  is  devoid 
of  this  constituent.  The  vast  accumulations  of  nitrogen,  known  to 
exist  in  all  arable  soils,  were  ascribed,  therefore,  to  the  residues  of  many 
generations  of  plants;  and  the  assumption  seemed  to  be  justified  that 
the  atmosphere,  79  per  cent  of  whose  bulk  consists  of  nitrogen  gas,  is 
the  direct  source  of  this  element  to  plants.  It  was  not  long,  however, 
before  plant  physiologists  demonstrated  experimentally  that  nitrogen 
gas  as  such  could  not  directly  serve  as  food  for  plants.  There  thus 
arose  one  of  the  most  interesting  and,  for  a  long  time,  one  of  the  most 
puzzling  problems  in  agricultural  research.  Among  the  earlier  in- 
vestigators de  Saussure  believed,  at  the  beginning  of  the  nineteenth 
century,  that  nitrogen  is  taken  up  from  the  soil  in  combined  form. 
Liebig  in  1840  advanced  his  well-known  "mineral  theory"  according 
to  which  plants  secured  their  nitrogen  from  the  air,  in  the  form  of 
ammonia.  He  assumed,  thus,  that  plants  cannot  use  elementary 
nitrogen,  and  that  the  supply  of  atmospheric  nitrogen  in  the  form  of 
ammonia  was  great  enough  to  meet  the  needs  of  growing  vegetation. 
The  latter  view  was  not  accepted  by  Lawes  and  Gilbert  of  the  Roth- 
amsted  Station  in  England.  By  a  series  of  elaborate  and  carefully 
controlled  experiments  they  demonstrated  in  1858  that  nitrogen  in  the 
elementary  form  cannot  be  used  by  plants.  They  further  demonstrated 
that  the  amount  of  combined  nitrogen  brought  down  in  the  form  of 
ammonia,  nitrites  and  nitrates,  by  atmospheric  precipitation  was  but 
slight  when  compared  with  the  quantities  annually  removed  by  crops. 
Hence  the  problem  as  to  the  source  and  maintenance  of  combined 
nitrogen  in  the  soil  seemed  to  be  more  puzzling  than  ever. 

400 


FIXATION   OF   ATMOSPHERIC   NITROGEN-  401 

CHEMICAL  AND  BIOLOGICAL  RELATIONS. — The  second  and  third 
quarters  of  the  nineteenth  century  saw  the  birth  of  a  number  of  theories 
dealing  with  this  problem.  It  was  suggested  that  nitrogen  compounds 
may  be  formed  in  the  soil  by  the  oxidation  of  nitrogen  to  nitric  acid. 
Compounds  of  iron,  manganese  and  lime  were  supposed  in  some  way 
to  make  such  oxidation  changes  possible.  It  was  likewise  suggested 
that  nascent  hydrogen  may  be  generated  in  the  decomposition  of  organic 
matter  in  the  soil,  and  reacting  with  elementary  nitrogen,  may  give 
rise  to  ammonia.  The  various  hypotheses  were  not  supported  by 
experimental  proof;  moreover,  the  situation  was  complicated  by  the 
knowledge,  based  on  empirical  observations,  that  crops  of  the  legume 
family  seemed  to  be  more  or  less  independent  of  the  supply  of  combined 
nitrogen  in  the  soil.  Indeed,  clovers  and  other  legumes  had,  appar- 
ently, the  ability  to  increase  the  content  of  combined  nitrogen  in  the 
soil  as  was  indicated  by  the  experiments  of  Boussingault  and  of  Lawes 
and  Gilbert.  .Finally,  the  mystery  was  solved  by  the  investigations 
of  Berthelot  and  Hellriegel  and  Wilfarth  who  furnished  the  proof  that 
elementary  nitrogen  may  be  utilized  by  plants  when  certain  biological 
relations  are  met.  These  relations  involve  the  presence  and  activities 
of  microorganisms  that  by  themselves,  or  in  conjunction  with  higher 
plants,  make  available  to  growing  vegetation  the  great  store  of 
atmospheric  nitrogen. 

NON-SYMBIOTIC  FIXATION  OF  NITROGEN 

HISTORICAL. — Non-symbiotic  nitrogen  fixation,  or  Azofication,  has 
already  been  defined  as  the  production  of  nitrogen  compounds  out  of 
atmospheric  nitrogen  by  bacteria  independently  of  higher  plants.  The 
part  played  by  bacteria  in  this  process  was  not  recognized  until  1885, 
when  Berthelot  published  some  of  his  data  on  the  accumulation  of  com- 
bined nitrogen  in  uncropped  soils.  His  results  seemed  to  explain  a 
number  of  scattered  observations,  made  since  the  middle  of  the  century, 
on  the  apparent  increase  of  the  nitrogen  content  of  cultivated  soils. 

While  Berthelot's  experiments  proved  that  the  nitrogen  gains 
occurred  only  in  unsterilized  soils  and  were,  therefore,  due  to  micro- 
organisms, it  remained  for  Winogradski  to  demonstrate,  in  1893,  that 
the  formation  of  nitrogen  compounds  by  certain  types  of  bacteria 
may  be  accomplished  in  culture  media  nearly  or  quite  devoid  of  com- 

: 


402  MICROBIOLOGY   OF   SOIL 

bined  nitrogen.  Soon  after  that  he  succeeded  in  isolating  his  organisms 
in  pure  culture,  and  described  them  as  anaerobic  bacilli  allied  to  those 
of  the  butyric  group.  In  1901  our  knowledge  of  Azobacteria  was 
enriched  by  Beyerinck's  discovery  of  a  group  of  large,  obligate  aerobic 
bacteria  that  he  designated  as  Azotobacter.  Since  that  date  it  has  been 
found  that  the  ability  to  fix  atmospheric  nitrogen  is  possessed  also  by 
certain  molds  and  by  various  species  of  bacteria.  However,  this  ability 
is  not  only  extremely  variable,  but  is  also  very  feeble  as  compared 
with  that  of  the  members  of  the  two  groups  described  by  Winogradski 
and  Beyerinck.  These  two  groups  may,  therefore,  be  designated  as 
including  the  nitrogen-fixing  bacteria  par  excellence. 

ANAEROBJC  SPECIES. — The  species  isolated  by  Winogradski  was 
named  by  him5.  (Clostridium)  pasteurianus  (Fig.  131).     It  was  found  to 


FIG.  131. — B.  (Clostridium)  pasteurianus,  a  non-symbiotic  nitrogen-fixing  organism. 
(After  Winogradski  from  Lipman.) 

grow  readily  under  anaerobic  conditions  in  culture  solutions  contain- 
ing dextrose  and  the  necessary  mineral  salts,  but  no  combined  nitrogen. 
The  products  of  growth  included  protein,  butyric  and  acetic  acids, 
carbon  dioxide  and  hydrogen.  In  the  presence  of  other  bacteria  B. 
(Clostridium)  pasteurianus  was  found  to  develop  also  under  aerobic 
conditions.  Subsequently  studies  by  Winogradski  and  other  investi- 
gators showed  that  B.  (Clostridium)  pasteurianus,  and  varieties  of  this 
species  are  very  widely  distributed  in  cultivated  soils.  More  recently 
Bredeman  made  a  thorough  and  extended  study  of  anaerobic  Azo- 
bacteria and  demonstrated  their  almost  invariable  presence  in  a  large 
number  of  soil  samples  from  Europe,  Asia  and  America.  In  his  opinion 
they  correspond  more  or  less  closely  to  B.  amylobacter  described  many 
years  before  by  van  Tieghem. 

AEROBIC  SPECIES. — A  more  or  less  pronounced  power  to  fix  atmos- 


FIXATION    OF   ATMOSPHERIC   NITROGEN  403 

pheric  nitrogen  is  apparently  possessed  by  a  considerable  number  of 
aerobic  species.  Lipman  has  demonstrated  the  fixation  of  small 
amounts  of  nitrogen  by  Ps.  pyocyanea  and  Lohnis  secured  similar  results 
with  Bad.  pneumonia,  B.  lactis  mscosus,  B.  radiobacter  and  B. 
prodigiosus.  Gottheil  has  detected  fixation  by  B.  ruminatus  and  B. 
simplex;  Pillai  has  described  a  nitrogen-fixing  aerobic  bacillus,  B. 
malabarensis;  Wester mann  studied  a  similar  organism  that  he  named  B. 
danicus;  while  Beyerinck  and  van  Delden  observed,  some  years  earlier, 
that,  certain  strains  of  B.  mesentericus  could  fix  relatively  large  amounts 
of  nitrogen.  Similarly  Ps.  radicicola  has  been  found  to  possess  a  slight, 
but  nevertheless  an  appreciable  power  to  fix  elementary  nitrogen  in 
culture  solutions  or  in  the  soil. 


FIG.  132. — Azotobacter  vinelandi,  a  non-symbiotic  nitrogen-fixing  organism. 
(After  Lipman.) 

But  while  nitrogen  fixation  among  aerobic  soil  bacteria  is  not  as 
uncommon  as  was  at  one  time  supposed,  this  function  is  so  feeble  and 
so  variable  in  most  instances,  as  to  be  of  negative,  or,  at  best,  of  doubt- 
ful economic  significance.  On  the  other  hand,  the  aerobic,  Azotobacter, 
first  described  by  Beyerinck  in  1901,  may  be  regarded  not  only  as  pos- 
sessing a  very  pronounced  ability  to  fix  atmospheric  nitrogen,  but  as 
playing  a  role  of  some  moment  in  maintaining  the  supply  of  combined 
nitrogen  in  the  soil. 

To  the  two  species  of  Azotobacter,  A.  chroococcum  and  A.  agilis 
described  by  Beyerinck  and  van  Delden,  Lipman  added  A.  mnelandii 


404  MICROBIOLOGY    OF    SOIL 

(Fig.  132),  A .  beyerincki  and  A .  woodstownii,  and  Lohnis  and  Westermann, 
A.  mtreum.  Of  these  species  A.  chroococcum  and  A.  beyerincki  are  most 
common  and  are  widely  distributed  in  cultivated  soils  of  Europe  and 
America,  and  probably  also  of  the  other  continents.  They  are  absent 
in  acid  soils  deficient  in  humus,  and  most  common  in  limestone  regions 
and  in  irrigated  soils  rich  in  mineral  salts.  Their  food  requirements  are 
covered  by  solutions  containing  potassium  phosphate,  magnesium 
sulphate,  calcium  chloride  and  ferric  sulphate,  and  some  organic 
nutrient,  such  as  dextrose,  saccharose,  xylose,  mannit,  acetate,  pro- 
pionate,  butyrate,  malate,  ethyl  alcohol,  etc.  An  alkaline  or  neutral 
reaction  and  the  presence  of  salts  of  iron  are  essential  for  the  vigorous 
development  of  Azotobacter,  while  humates  have  been  shown  by 
Krzemieniewski  to  exert  a  stimulating  influence  on  the  growth  of  these 
organisms,  even  though  not  acting  directly  as  a  source  of  food  and 
energy.  As  shown  by  Lipman  and  others,  Azotobacter  may  gain  an 
increased  power  of  fixing  atmospheric  nitrogen  in  the  presence  of  other 
organisms.  It  is  resistant  to  drying,  notwithstanding  the  fact  that  it 
produces  no  spores,  and  has  been  successfully  isolated  from  soil  samples 
that  had  been  kept  in  a  dry  state  for  several  years.  For  some  reason 
it  may  be  detected  in  the  soil  most  readily  in  the  fall  and  winter 
months. 

As  to  the  nitrogen-fixation  by  fungi,  it  has  been  shown  elsewhere 
that  the  evidence  is,  if  anything,  of  a  negative  character.  Some 
algae  are  able  to  fix  atmospheric  nitrogen,  especially  those  that  live 
symbiotically  with  azotobacter. 

ENERGY  RELATIONS. — In  the  fixation  of  nitrogen  by  bacteria  the 
necessary  energy  for  the  process  is  furnished  by  the  carbohydrates, 
organic  acids,  alcohols  or  other  organic  nutrients  employed  in  the 
culture  media.  Since  any  given  quantity  of  organic  nutrient  possesses 
a  definite  amount  of  potential  energy  the  fixation  of  nitrogen  is  neces- 
sarily limited  by  the  supply  of  such  potential  energy.  This  limitation 
was  already  recognized  by  Winogradski  in  his  experiments  with  B. 
(Clostridium)  pasteurianus .  For  every  gram  of  dextrose  used  up  there 
was  produced,  on  the  average,  2  to  3  mg.  of  combined  nitrogen.  In  the 
experiments  of  Bredeman  with  B.  amylobacter,  and  of  Pringsheim  with 
11  Clostridium  americanum"  the  amounts  fixed  were,  at  times,  con- 
siderably larger.  On  the  whole,  however,  it  has  been  proved  by  a 
number  of  investigators  that  Azotobacter  can  fix  much  larger  quantities 


FIXATION    OF    ATMOSPHERIC    NITROGEN  405 

of  nitrogen  than  the  anaerobic  bacilli.  The  extended  investigations 
of  Lipman  showed  that  A .  vinelandii  has  the  ability  to  fix  more  nitrogen 
per  unit  of  organic  nutrient  consumed  than  either  A.  chroococcum  or 
A .  beyerincki.  Under  favorable  conditions  A .  vinelandii  may  at  times 
fix  15  or  even  20  mg.  of  nitrogen  per  g.  of  mannit  used  up.  Krze- 
mieniewski  found  in  experiments  with  A.  chroococcum  that  additions 
of  humates  to  the  culture  solutions  increased  the  nitrogen  fixed  from  a 
maximum  of  2.4  mg.  to  a  maximum  of  14.9  mg. 

The  practical  bearing  of  the  foregoing  data  lies  in  the  fact  that  the 
fixation  of  nitrogen  in  cultivated  soils  is  limited,  among  other  things,  by 
the  energy  available,  that  is,  by  the  quantity  of  readily  decomposable 
organic  residues.  An  indication  as  to  the  extent  of  these  is  given  by  the 
amount  of  humus  present;  nevertheless,  this  must  remain  an  indication 
merely,  for  most  of  the  humus  is  too  inert  to  serve  as  a  source  of  energy 
to  Azotobacter.  From  the  data  at  present  available  different  investi- 
gators have  estimated  the  quantity  of  nitrogen  fixed  by  Azotobacter 
at  6.8  kg.  to  18  kg.  (15  to  40  pounds)  per  acre,  per  annum.  Assuming 
favorable  conditions  for  fixation,  so  that  500  g.  (i  pound)  of  nitrogen 
could  be  fixed  for  every  50  kg.(ioo  pounds)  of  carbohydrate  consumed, 
it  would  still  take  an  equivalent  of  680  kg.  to  1,814  kg.  (1,500  to  4,000 
pounds)  of  sugar  to  produce  this  quantity  of  combined  nitrogen.  It  may 
be  noted  in  this  connection  that  Azotobacter  have  been  demonstrated 
to  live  in  symbiosis  with  algae,  obtaining  thereby  the  necessary  energy 
for  their  activities.  This  may  explain,  perhaps,  the  remarkable  facts 
observed  by  Headden  in  Colorado,  relating  to  the  accumulation  of  such 
enormous  quantities  of  nitrate  in  the  soil  as  to  destroy  all  vegetation. 
In  some  instances  the  nitrates  were  found  to  be  present  to  the  extent  of 
90,718  kg.  (100  tons),  or  more  (per  acre),  to  a  depth  of  a  few  inches.  If 
the  accumulation  of  combined  nitrogen  was  due  to  Azotobacter,  as  is 
claimed  by  Headden,  and  the  bacterial  residues  oxidized  by  nitrifying 
bacteria  to  nitrates,  it  is  difficult  to  account  for  the  source  of  the  1,000 
or  2,000  tons  of  carbohydrates  necessarily  used  up  in  the  process  of 
fixation,  unless  it  could  be  proved  that  the  energy  was  furnished  by 
algae. 

SYMBIOTIC  FIXATION 

HISTORICAL. — Empirical  observations  extending  well  back  into 
ancient  agriculture  have  led  to  the  recognition  of  the  soil-enriching 


406  MICROBIOLOGY   OF    SOIL 

qualities  of  certain  crops  of  the  legume  family.  Columella  mentions 
the  fact  that  many  Roman  farmers  regarded  beans  as  possessing  these 
qualities,  but  does  not  accept  this  belief  for  himself.  On  the  other 
hand,  he  points  out  that  luzerne  (alfalfa),  lupins  and  vetches  improve 
the  land  and  act  as  manure.  He  points  out,  also,  that  it  was  the 
practice  of  Roman  farmers  to  plow  under  lupines  in  order  to  enrich  the 
soil.  In  the  centuries  following  the  fall  of  Rome  the  use  of  legumes  for 
soil  improvement  persisted  to  some  extent  in  Italy,  France  and  other 
countries;  yet  the  practice  was  not  followed  consistently  and  the  fer- 
tility of  European  soils  was  declining  for  lack  of  available  nitrogen, 
and,  to  a  large  extent,  also  of  phosphoric  acid.  The  more  general  intro- 
duction of  clover  into  Germany  and  England  in  the  eighteenth  century 
helped  to  restore  the  fertility  of  many  farms,  and  led,  ultimately,  to  the 
recognition  of  the  peculiar  place  held  by  legumes  in  the  maintenance 
of  soil  fertility.  But  while  practical  farmers  knew  of  the  soil-enriching 
power  of  legumes,  and  while  they  retained  their  belief  in  it  even  when 
it  seemed  contrary  to  scientific  authority,  they  did  not  know  the  secret 
of  this  power.  It  remained  for  Hellriegel  and  Wilfarth  to  demonstrate 
in  1886,  and  more  fully  in  1888,  that  this  power,  already  hinted  at  by 
the  investigations  of  others,  is  the  resultant  of  the  combined  activities 
of  the  plants  and  of  bacteria  that  enter  their  roots,  and  produce  there 
the  well-known  nodules  or  tubercles.  They  showed  in  no  uncertain 
manner  that  legumes  can  improve  the  soil  only  in  so  far  as  they  add 
nitrogen  to  it  with  the  aid  of  the  bacteria  in  the  tubercles;  in  other 
words,  legumes  were  shown  to  enter  into  a  symbiotic  relationship  with 
certain  bacteria  and  to  acquire,  thereby,  the  ability  to  fix  atmospheric 
nitrogen. 

The  presence  of  tubercles  on  the  roots  of  leguminous  plants  was  first 
recorded  by  Malpighi  in  1687.  He  regarded  them  as  root  galls.  The 
botanists  who  studied  them  in  the  first  half  of  the  nineteenth  century 
classified  them  as  modifications  of  normal  roots  or  as  pathological 
processes.  In  1866  the  Russian  botanist  Woronin  found  that  the 
tubercles  were  filled  with  minute  bodies  resembling  bacteria  and  con- 
cluded that  they  were  pathological  outgrowths.  Some  years  later 
Frank,  in  1879,  not  onty  showed  that  tubercles  are  almost  invariably 
present  on  the  roots  of  legumes,  but  that  their  formation  may  be  pre- 
vented by  sterilizing  the  soil.  Frank  was  thus  in  possession  of  facts 
that  might  have  revealed  to  him  the  true  nature  of  the  root-tubercles. 


FIXATION   OF   ATMOSPHERIC   NITROGEN  407 

However,  he  later  modified  his  belief  in  the  origin  of  tubercles  as  due 
to  outside  infection,  and  accepted  the  interpretation  of  his  pupil 
Brunchhorst  who  claimed  that  the  bacteria-like  bodies  in  the  tubercles 
were  merely  reserve  food  materials.  Because  of  their  resemblance  to 
bacteria  Brunchhorst  named  them  bacteroids. 

The  studies  of  Marshall  Ward,  published  in  1887,  proved  not  merely 
that  tubercle  formation  is  due  to  outside  infection,  but  that  such  infec- 
tion may  be  brought  about  at  will  by  placing  the  roots  of  young  plants 
in  contact  with  pieces  of  old  tubercles.  Hellriegel  in  his  preliminary 
communication  of  1886  also  showed  that  outside  infection  is  necessary 
for  the  production  of  tubercles,  and  called  attention  to  the  true  func- 


FIG.  133.— Ps.  radidcoia.     i,  From  Melilotus  alba;  2  and  3,  from  Medicago  saliva. 
4,  from  Vicia  mllosa.     (After  Harrison  and  Barlow  from  Lipman.) 

tion  of  the  latter  as  laboratories  wherein  nitrogen  compounds  are 
manufactured  out  of  elementary  nitrogen.  The  true  worth  of  Hell- 
riegel's  investigations  was  brought  out  more  clearly  in  another  paper 
that  he  published  jointly  with  Wilfarth  in  1888.  The  authors  showed 
that  in  sterilized  soils  legumes  behaved  precisely  like  non-legumes,  and 
died  ultimately  of  nitrogen  hunger  when  not  provided  with  nitrates  or 
other  suitable  nitrogen  compounds.  On  the  other  hand,  when  the 
sterilized  soil  was  later  infected  with  a  few  drops  of  leachings  from  fresh 
soil  that  had  supported  a  normal  growth  of  legumes,  the  starving  plants 
recovered  and  grew  vigorously.  Under  the  same  conditions  non- 
legumes  did  not  recover.  The  recovery  of  the  starving  legumes  was 
found  to  be  coincident  with  the  formation  of  tubercles. 


4o8 


MICROBIOLOGY    OF    SOIL 


Hellriegel  and  Wilfarth's  studies  were  soon  confirmed  by  the  inves- 
tigations of  others.  Wigand  showed  in  1887  that  the  tubercles  con- 
tained within  them  true  bacteria.  In  the  following  year  Beyerinck 
reported  the  successful  isolation  of  these  bacteria  on  artificial  media, 
and  named  the  organism  B.  radicicola  (Fig.  133).  Prazmowski  also 
isolated  pure  cultures  of  Ps.  radicicola,  and  followed  the  entrance  of 
the  organisms  into  the  root  hairs  of  young  plants,  their  passage  through 
the  cell- walls,  and  their  transformation  into  bacteroids.  These  facts 
were  all  confirmed  by  other  investigators,  and  it  was  further  shown  by 
Schloesing  and  Laurent  that  properly  inoculated  legumes  not  only  can 
grow  in  soils  devoid  of  combined  nitrogen,  but  that  when  growing  in 
such  soils  in  a  confined  atmosphere  they  decrease  the  quantity  of 
nitrogen  gas  surrounding  them  by  transforming  it  into  nitrogen  com- 
pounds. It  was,  therefore,  made  clear  by  these  investigations,  and  by 


FIG.  134. — Sections  through  root  tubercles,  i,  Cell  from  tubercle  of  Pisum 
sativum,  snowing  bacterial  filament;  2  and  3,  cells  with  bacterial  filaments  from 
tubercle  of  Trifolium  pannonicum.  (After  Stefan  from  Lipman.) 

others  not  mentioned  here  for  lack  of  space,  that  the  belief  of  practical 
farmers  in  the  soil  enriching  qualities  of  legumes  was  amply  justified. 
It  was  shown,  further,  that  the  later  experiments  of  Boussingault,  as 
well  as  those  of  Lawes,  Gilbert  and  Pugh  failed  to  solve  the  problem 
because  these  investigators  treated  their  soil  so  as  to  prevent  the 
survival  and  subsequent  entrance  of  Ps.  radicicola,  and  deprived  the 
leguminous  plants  of  the  ability  to  utilize  atmospheric  nitrogen. 

MODES  or  ENTRANCE  AND  DEVELOPMENT. — Tubercle  bacteria  con- 
sisting of  small  motile  rods  usually  enter  the  legumes  by  way  of  the  root- 
hairs.  For  this  reason  young  tubercles,  with  but  few  exceptions,  are 
found  on  young  roots.  The  organisms  multiply  at  the  point  of  infection 
and  penetrate  into  adjacent  plant-tissue  by  means  of  a  hypha-like 


^FIXATION   OF   ATMOSPHERIC    NITROGEN  409 

hollow  thread  or  tube  that  seems  to  consist  of  a  gelatinous  material 
(Fig.  134) .  The  tubes  branch  out  as  they  pass  from  cell  to  cell  and  carry 
the  invading  organisms  with  them.  The  bacteria  which  may  be  readily 
detected  within  the  tubes  and  cells  are  the  involution  forms  of  Ps. 
radicicola  and  assume  various  irregular  shapes.  They  are  designated 
as  bacteroids.  Stefan  has  suggested  that  bacteroids  may  be  produced 
within  the  tubes  and,  possibly,  from  the  buds  or  swellings  that  appear 
on  the  tubes.  While  still  young,  the  bacteroids  are  capable  of  dividing, 
but  as  they  grow  they  swell  up  and  finally  degenerate. 

RESISTANCE,  IMMUNITY  AND  PHYSIOLOGICAL  EFFICIENCY. — The 
invasion  of  legumes  by  Ps.  radicicola  and  the  acquisition  by  the  plant, 
thanks  to  this  invasion,  of  the  power  to  fix  elementary  nitrogen  are  cited 
as  a  case  of  symbiosis.  However,  some  writers  would  regard  the  pres- 
ence of  Ps.  radicicola  in  legume  roots  as  a  case  of  parasitism.  According 
to  them  symbiosis  presupposes  the  living  together  of  two  organisms 
with  resulting  benefit  to  both.  In  the  present  instance,  however, 
conditions  may  arise  when  the  host  plant  is  injured,  rather  than  bene- 
fited; and  similarly,  conditions  may  arise  when  the  invading  bacteria 
are  suppressed  by  the  plants.  Making  due  allowance  for  the  ob- 
jections raised  we  still  find,  nevertheless,  that  in  the  broad  relation  of 
the  two  groups  of  organisms  there  is  an  apparent  benefit  to  both  plants 
and  bacteria.  The  former  gain  an  adequate  supply  of  nitrogen  and 
the  latter  a  supply  of  carbohydrates  and  of  mineral  salts. 

A  more  detailed  study  of  this  relation  shows  that  the  plants  resist 
the  entrance  of  bacteria.  When  an  abundance  of  available  nitrogen 
compounds  is  supplied  tubercle  formation  may  be  largely  or  wholly 
suppressed.  In  that  case  the  plants  secure  their  nitrogen  from  the  soil 
and  are  not  only  independent  of  the  bacteria,  but  are  strong  enough  to 
resist  their  entrance.  It  is  further  claimed  by  Hiltner  that  tubercle 
bacteria  differ  in  their  virulence,  and  that  the  more  virulent  the  organ- 
isms, the  more  readily  will  they  penetrate  the  root  tissue.  Moreover, 
he  believes  that  when  a  plant  is  invaded  by  organisms  of  any  degree  of 
virulence,  the  host  plant  becomes  immune  to  a  large  extent  and  can  keep 
out  all  but  the  most  virulent  bacteria.  The  use  of  the  term  virulence, 
in  this  connection,  has  been  objected  to,  since  it  is  borrowed  from 
animal  pathology  and  is  likely  to  be  misleading.  It  is  better  to  employ 
the  term  physiological  efficiency  as  implying  not  only  a  more  pro- 
nounced ability  to  enter  the  plant  roots,  but  also  to  fix  atmospheric 


410  MICROBIOLOGY   OF    SOIL 

• 

nitrogen.  It  is  conceivable  that  strains  of  Ps.  radicicola  may.be  de- 
veloped that  would  grow  rapidly  and  yet  possess  but  a  feeble  nitrogen- 
fixing  power.  In  other  words,  they  would  possess  a  high  vegetative 
power  and  a  low  physiological  efficiency. 

MECHANISM  or  FIXATION. — It  is  generally  believed  that  the  fixation 
of  nitrogen  is  accomplished  by  the  bacteria  within  the  tubercles.  The 
claim,  at  one  time,  advanced  by  Stoklasa,  that  the  fixation  is  accom- 
plished by  the  plants  themselves  with  the  aid  of  enzymes  produced  by 
the  bacteria  in  their  roots,  has  been  disproved.  It  is  known  that  the 
period  of  active  nitrogen  assimilation  by  the  plants  coincides  with  the 
appearance  of  the  bacteroids  in  the  tubercles,  and  it  is  supposed  that 
the  microorganisms  fashion  nitrogen  compounds  out  of  atmospheric 
nitrogen  by  using  the  carbohydrates  and  organic  acids  in  the  plant 
juices  as  a  source  of  energy.  The  plants  then  seem  to  utilize  the  soluble 
nitrogen  compounds  that  pass  out  of  the  bacterial  cells.  It  is  further 
supposed  that  bacteroid  formation  is  an  attempt  on  the  part  of  the 
microorganisms  to  adjust  themselves  to  the  drain  caused  by  the 
activities  of  the  host  plant. 

VARIATIONS  AND  SPECIALIZATION. — Apparent  differences  in  bacteria 
from  different  legumes  were  noted  by  Hellriegel.  Some  of  his  experi- 
ments indicated  that  bacteria  from  clovers  could  not  produce  tubercles 
on  lupines  and  serradella.  Analogous  differences  were  found  by 
Nobbe  and  his  associates,  nevertheless  they  were  finally  led  to  conclude 
that  the  root  invasion  of  legumes  is  caused  by  a  single  species.  How- 
ever, continued  association  with  any  particular  legume  accomplished 
in  the  end  a  certain  modification,  or  specialization,  as  it  were,  of  the 
microorganisms,  and  they  were  then  no  longer  able  to  invade  the  roots 
of  other  legumes.  Later,  Hiltner  and  Stormer  have  been  led  to 
modify  this  view  and  have  arranged  the  tubercle  bacteria  in  two 
groups,  possessing,  according  to  them,  well-defined  morphological  and 
physiological  differences.  One  of  these  groups  is  included  under  the 
species  " Rhizobium  radicicola"  and  the  other  under  " Rhizobium 
beyerinckii."  The  former  comprises  the  organisms  from  lupines,  serra- 
della and  soy  beans  while  the  latter  comprises  all  of  the  others. 

RELATION  TO  ENVIRONMENT. — Nitrogen  fixation  by  leguminous 
vegetation  is  readily  influenced  by  soil  conditions,  particularly  the 
supply  of  lime  and  of  other  basic  substances;  the  supply  of  organic 
matter  and  the  aeration  of  the  soil.  As  to  the  first  of  these  it  is  well 


FIXATION   OF   ATMOSPHERIC   NITROGEN 


411 


known  that  all  legumes,  with  the  exception  of  lupines  and  serradella, 
are  stimulated  in  their    growth  by    generous    applications  of  lime. 


FIG.  135. — These  two  pea  plants  were  grown  in  clean  quartz  sand  to  which  had 
been  added  small  quantities  of  all  the  necessary  elements  of  plant  food  except 
nitrogen.  The  conditions  were  exactly  identical  except  that  plant  A  was  without 
root  nodules  (see  Fig.  136)  and  plant  B  had  numerous  nodules  well  developed  (see 
Fig.  137).  (Mich.  Exp.  Station.) 


The  top  dressing  of  lawns  with  lime,  marl  or  wood  ashes  encourages 
the  appearance  of  white  clover;  an  adequate  supply  of  lime  makes 


412 


MICROBIOLOGY   OF    SOIL 


possible  the  successful  growing  of  alfalfa  in  almost  any  soil,  while  the 
leguminous  vegetation  of  limestone  soils  is  proverbially  vigorous. 
The  favorable  influence  of  lime  is  due  to  the  direct  action  on  the  plants 
as  well  as  on  the  bacteria  in  the  soil.  Similarly,  the  tubercle  bacteria 
are  favorably  affected  in  their  survival  and  multiplication  by  an 
abundant  supply  of  organic  matter.  On  the  other  hand,  acid  soils  or 
those  deficient  in  humus  and  inadequately  aerated  are  but  ill  suited 
to  the  activities  of  Ps.  radicicola. 


FIG.  136. — Roots  of  Plant  A  without  nodules  (Fig.  135). 


SOIL  INOCULATION* 

By  soil  inoculation  is  now  understood  the  adoption  of  some 
artificial  method  for  supplying  suitable  quantities  of  nitrogen-fixing 
organisms  to  soils  deficient  in  these  types.  The  first  attempts  at  soil 
inoculation  were  made  in  1886  by  Hellriegel  and  Wilfarth  during  the 

*  Prepared  by  S.  F.  Edwards. 


FIXATION   OF   ATMOSPHERIC   NITROGEN  413 

course  of  their  studies  on  the  cause  of  nitrogen  accumulation  by 
legumes.  They  found  that  when  leguminous  plants  were  grown  in 
sterile  sand,  nodules  were  formed  on  the  roots  only  after  the  addition 
of  a  small  portion  of  aqueous  extract  of  fertile  soil,  or  an  extract  of 
crushed  nodules,  or  in  some  cases  (lupines  and  seradella)  by  soil  itself 
from  a  field  on  which  these  crops  had  been  grown.  The  first  successful 
artificial  production  of  nodules  by  the  aid  of  pure  cultures  was  made 


FIG.  137. — Roots  of  plant  B  with  nodules  (Fig.  135). 

in  1889  by  Prazmowski  in  the  course  of  studies  on  the  method  of 
entrance  of  the  organism  to  the  root  hairs  of  the  host  plant. 

The  first  inoculation  experiments  in  a  large  way  were  those  made  in 
1887  at  the  Moor  Soil  Experiment  Station,  Bremen,  Germany,  where 
earth  taken  from  fields  that  had  borne  luxuriant  crops  of  various 
legumes  was  scattered  over  reclaimed  heath  or  swamp  soils  upon  which 
legumes  had  not  previously  grown,  with  the  result  that  in  every  instance 
the  yield  on  the  inoculated  portions  of  land  was  greater  than  on  the 


414  MICROBIOLOGY   OF   SOIL 

uninoculated  plots.  After  such  favorable  results,  it  was  but  a  natural 
step  to  try  the  effect  of  similar  applications  of  soil  rich  in  the  nodule- 
forming  bacteria  to  ordinary  cultivated  soils  of  varying  character. 
While  results  in  some  cases  were  eminently  satisfactory,  in  others  there 
was  no  increase  in  the  vigor  or  amount  of  the  crop  as  a  result  of  the 
inoculation. 

METHODS  or  SOIL  INOCULATION. — From  these  early  experimental 
results  there  evolved  two  general  methods  of  inoculation,  namely,  the 
application  of  soil  from  an  already  inoculated  field,  and  the  application 
of  pure  cultures  of  the  nodule-forming  bacteria  to  the  seed  before 
sowing. 

Inoculation  with  Legume-earth. — The  use  of  soil  as  inoculating 
material  -was  tried  by  various  experiment  stations  of  the  United  States, 
with  results  not  varying  widely  from  those  secured  in  the  pioneer 
experimental  work  at  Bremen.  It  was  found  in  general  that  the 
commonly  grown  crops,  such  as  the  common  clovers,  peas  and  beans, 
made  little  or  no  increase  as  a  result  of  inoculation  with  old  legume-soil. 
With  new  crops,  however,  such  as  alfalfa  and  soy  beans  when  they  were 
first  introduced,  it  was  found  impossible  in  many  places  to  secure  a 
successful  stand  until  the  fields  on  which  these  crops  were  to  be  grown 
had  received  a  top-dressing  of  soil  from  land  that  had  already  grown 
the  crop  in  question;  and  it  became  a  common  practice  to  inoculate 
soil  in  this  manner  before  seeding  with  these  new  crops.  It  was  early 
observed,  however,  that  this  method  of  soil  transfer  for  inoculation 
purposes  was  not  an  unmixed  benefit.  Aside  from  the  expense  and 
difficulty  of  handling  and  transportation  of  soil,  fungus  and  bacterial 
diseases,  not  only  of  legumes  but  of  other  crops,  as  well  as  the  seeds 
of  noxious  weeds,  were  transmitted  from  one  field  to  another  and  even 
from  one  section  of  country  to  another.  It  was  to  avoid  this  difficulty 
that  the  preparation  of  pure  cultures  was  introduced. 

Inoculation  with  Pure  Cultures.  Nitragin. — The  first  pure  culture 
method  was  launched  in  1896  by  Nobbe  and  Hiltner,  German  investi- 
gators, who  prepared  cultures  of  the  legume  bacteria  on  nutrient  gelatin 
and  arranged  with  a  firm  of  manufacturing  chemists  to  place  them  on 
the  market  under  the  trade  name  of  'Nitragin. 

Dried  Cultures. — In  the  United  States  the  matter  of  pure  cultures 
was  first  taken  up  by  the  Department  of  Agriculture  about  1902. 
Cultures  of  the  nodule-forming  bacteria  were  cultivated  in  nitrogen- 


FIXATION    OF    ATMOSPHERIC    NITROGEN  415 

free  culture  media,  dried  on  cotton  and  distributed  to  farmers  with  a 
small  package  of  salts  from  which  a  culture  solution  was  to  be  made 
by  the  farmer  and  applied  to  the  seed.  This  method  gave  poor  results, 
chiefly  because  the  bacteria  could  not  withstand  the  drying  on  cotton. 
Afterward  the  cultures  were  sent  in  a  liquid  condition  with  somewhat 
more  satisfactory  results.  The  dry  cotton  cultures  were  exploited 
for  a  time  by  a  commercial  firm  under  the  name  of  Nitro-culture,  and 
somewhat  similar  cultures  were  placed  on  the  market  in  England  under 
the  name  of  Nitro-bacterine.  Cultures  of  both  kinds,  however,  were 
shown  to  be  valueless,  both  by  microbiological  and  by  planting  tests. 

Cultures  on  A  gar. — Very  satisfactory  results  were  secured  from  the 
use  of  pure  cultures  at  the  Ontario  Agricultural  College,  Guelph,  where 
Harrison  and  Barlow,  in  1905,  originated  the  method  of  growing  the 
bacteria  on  a  nitrogen-poor  agar  medium.  By  this  method,  the  farmer 
has  simply  to  apply  the  bacteria  to  the  seed  just  before  sowing.  These 
cultures,  used  on  all  the  common  legumes,  sown  in  all  kinds  of  soil, 
gave  favorable  results  in  65  per  cent  of  cases  in  trials  extending  over  a 
period  of  ten  years.  Similar  agar  cultures  are  now  prepared  by  com- 
mercial firms  who  have  adopted  the  method  of  Harrison  and  Barlow, 
and  also  by  some  of  the  U.  S.  Agricultural  Experiment  Stations. 

Cultures  in  Soil.* — Temple  has  suggested  that  sterilized  soil  with 
the  addition  of  a  small  amount  of  leguminous  material  furnished  a 
very  good  medium  for  the  propagation  of  legume  bacteria  and  is  suitable 
for  their  distribution. 

Attempts  have  been  made  to  put  on  the  market  cultures  containing 
so-called  "fertilizing  bacteria"  good  for  "all  crops,"  but  the  tests  made 
with  these  cultures  have  thus  far  failed  to  bear  out  the  claims  made  for 
them.  The  successful  commercial  exploitation  of  cultures  containing 
strong  cellulose  and  protein  decomposing  organisms,  non-symbiotic 
nitrogen-fixing  organisms,  strong  nitrifying  organisms  and  other  useful 
bacteria  is  still  to  be  accomplished. 

Importance  of  Inoculation. — Inoculation  with  pure  cultures  affords 
the  farmer  a  rapid,  easy,  and  cheap  method  of  supplying  the  bacteria 
essential  for  getting  a  successful  stand  of  any  legumes.  Failure  to  secure 
a  benefit  from  this  method  of  inoculation  may  usually  be  attributed  to 
unsuitable  soil  conditions  rather  than  any  inherent  failing  in  the  cul- 
tures used.  No  method  of  inoculation  will  compensate  for  poor 

*  Prepared  by  Jacob  G.  Lipman. 


41 6  MICROBIOLOGY   OF    SOIL 

physical  or  chemical  condition  of  the  soil  itself.  The  principle  of  using 
artificial  cultures  to  be  applied  with  the  seed  is  sound,  and  if  the  cul- 
tures contain  large  numbers  of  virile  bacteria,  there  is  little  reason 
why  they  should  not  prove  of  benefit  when  used  under  soil  conditions 
that  would  seem  to  need  inoculation. 

Azotobacter  Cultures. — Some  experimental  work  has  been  done  in 
the  use  of  cultures  of  Azotobacter  for  soil  inoculation.  The  results  are 
contradictory,  and  more  work  needs  to  be  done  to  prove  the  value 
of  such  cultures. 


CHAPTER  IV 

CHANGES  IN  INORGANIC  CONSTITUENTS 
WEATHERING  PROCESS 

ORIGIN  AND  FORMATION  or  SOIL. — Rock  surfaces  exposed  to  the 
action  of  rain,  sunshine  and  frost  lose  their  fresh  appearance,  become 
pitted  and  uneven,  and  gradually  crumble  into  larger  and  smaller  frag- 
ments. In  the  course  of  time  the  layer  of  disintegrated  material 
becomes  deeper  and  its  constituent  particles  smaller — thanks  to  the 
uninterrupted  process  of  subdivision.  Finally,  lichens,  algae  and 
bacteria  make  their  appearance,  the  organic  debris  accumulates,  and 
higher  plants  begin  to  find  a  suitable  environment  for  their  development, 
The  rock  has  changed  into  soil. 

INFLUENCE  OF  BIOLOGICAL  FACTORS. — Soil-formation  is  not  entirely 
a  mechanical  or  chemical  process.  Even  before  the  layer  of  weathered 
rock  acquires  any  appreciable  depth  microscopical  and  macroscopical 
forms  of  life  gain  a  foothold  on  the  uneven  surface.  With  the  aid  of 
sunlight  they  build  organic  compounds  and  make  use  of  the  combined  or 
elementary  nitrogen  of  the  atmosphere.  Their  life  activities  result  in 
the  production  of  carbon  dioxide  and  of  varying  organic  and  inorganic 
acids  which  in  their  turn  react  with  the  constituents  of  the  rock  par  tides. 
In  this  manner  the  biological  activities  become  of  utmost  moment  in 
the  transformation  and  migration  of  mineral  substances  in  nature. 
They  assume  an  important  role  in  the  circulation  of  calcium  and  mag- 
nesium, with  the  accompanying  phenomena  that  find  most  striking 
expression  in  the  formation  of  caves  and  canyons  in  limestone  strata. 
They  assume  a  no  less  important  role  in  the  circulation  of  sulphur; 
in  the  accumulation  and  removal  of  available  potash  compounds  in 
the  soil,  as  well  as  in  the  transformation  of  phosphorus  and  its  migration 
from  inorganic  to  organic  compounds. 

LIME  AND  MAGNESIA 

REMOVAL  AND  REGENERATION  OF  CARBONATES.— Lime  and  mag- 
nesia are  present  in  soils  in  different  combinations.    They  may  occur 
27  417 


418  MICROBIOLOGY    OF    SOIL 

as  silicates,  carbonates,  phosphates,  humates,  sulphates,  etc.  In 
humid  climates  the  carbonates  are  being  continually  removed  from 
weathered  rock  material,  as  is  plainly  shown  by  the  composition  of 
drainage  waters.  The  losses  become  much  greater  in  cultivated  soils — 
thanks  to  the  humus  and  the  microorganisms  present  in  them.  The 
absolute  amounts  lost  from  year  to  year  will  depend  on  the  proportion 
of  lime  and  magnesia  in  the  soil,  the  mechanical  composition  of  the 
latter,  its  content  of  humus  and  the  methods  of  tillage  and  fertilization. 
According  to  Hall  the  soils  of  the.  experiment  fields  at  Rothamsted, 
containing  about  3  per  cent  of  calcium  carbonate,  are  losing  lime  at  the 
rate  of  362  kg.  to  453  kg.  (800  to  1,000  pounds)  per  acre  annually.  In 
certain  sections  of  Scotland  where  liming  has  been  practised  for  a  long 
time  the  farmers  estimate  the  loss  of  lime  from  the  land  at  6  bushels 
per  acre,  annually;  that  is,  approximately  at  the  rate  of  226  kg.  to 
272  kg.  (500  to  600  pounds).  In  New  Jersey,  New  York,  Pennsylvania 
and  other  eastern  states  farmers  who  use  lime  more  or  less  regularly 
apply  i  ton  of  it  at  the  beginning  of  each  five-year  rotation.  This  would 
provide  for  an  annual  loss  of  181  kg.  (400  pounds)  per  acre.  The  loss  of 
lime  and  magnesia  is  increased  under  intensive  methods  of  agriculture. 
When  animal  manures  and  green  manures  are  employed,  microbial 
activities  are  stimulated,  the  production  of  carbon  dioxide  is  encouraged 
and  the  loss  of  the  soluble  calcium  bicarbonate  made  greater.  The 
removal  of  lime  is  hastened  even  to  a  more  striking  extent  when 
ammonium  salts  are  applied  to  the  land.  The  resulting  nitrification 
and  loss  of  lime  are  illustrated  by  the  following  equation: 

(NH4)2SO4  +  2CaCO3  +  4O2  =  Ca(NO3)2  +  CaSO4  +  4H2O+2CO2 

As  was  already  indicated,  the  loss  of  calcium  and  magnesium  car- 
bonate from  the  soil  is  effected  largely  through  the  activities  of  bacteria 
and  of  other  microorganisms.  At  the  same  time  microorganic  life  is 
responsible  for  the  restoration  of  varying  amounts  of  carbonates.  It 
has  been  demonstrated  that,  in  the  weathering  of  the  complex  silicates, 
carbonates  and  silicic  acid  may  be  formed  in  considerable  quantities. 
In  the  presence  of  decaying  organic  matter  and  the  consequent  evolu- 
tion of  carbon  dioxide  the  formation  of  carbonates  from  silicates  may 
be  extensive  enough  to  balance  the  losses.  Similarly,  calcium  carbonate 
may  be  formed  in  the  soil  from  humates  and  from  the  calcium  salts  of 
simpler  organic  acids.  They  may  be  formed,  also,  through  the  activi- 


CHANGES    IN   INORGANIC   CONSTITUENTS  419 

ties  of  denitrifying  and  other  reducing  bacteria  from  the  corresponding 
nitrates  and  sulphates.  As  pointed  out  by  Nadson  ammonium  car- 
bonate produced  in  the  decomposition  of  protein  compounds  may  react 
with  calcium  sulphate  as  follows: 

COj  +  CaS04  =  CaCO3  +  (NH4)2SO4 


Moreover,  calcium  sulphate  may  be  reduced  to  sulphide  and  may  react 
with  carbon  dioxide  as  follows: 

CaS  +  CO2  +  H2O  =  CaCO3  +  H2S 

Magnesium  would  be  subject  to  similar  reactions  and  Nadson  has 
observed  the  formation  of  a  mixture  of  calcium  and  magnesium  car- 
bonates (corresponding  to  dolomite  in  composition)  in  media  inoculated 
with  a  pure  culture  of  B.  (Proteus)  vulgaris. 

LIME  AS  A  BASE.  —  The  carbon  dioxide  generated  in  vast  amounts  in 
the  life  processes  of  most  soil  bacteria,  the  nitrous  and  nitric  acids 
formed  by  the  nitro-bacteria,  the  sulphuric  acid  produced  in  the 
oxidation  of  hydrogen  sulphide  and  of  sulphur  by  the  so-called  sulphur 
bacteria,  and  the  great  variety  of  organic  acids  formed  in  the  decom- 
position of  carbohydrates,  fats  and  proteins  all  react  with  basic  sub- 
stances in  the  soil.  Of  these  basic  substances  calcium  carbonate  is  by 
far  the  most  prominent.  Combining  with  the  different  acids  it 
maintains  a  favorable  reaction  for  microorganic  life  in  the  soil. 

The  calcium  salts  thus  formed  are  more  or  less  soluble.  In  this 
manner  enormous  amounts  of  lime  are  annually  carried  to  the  ocean 
as  bicarbonate,  and  to  an  appreciable  extent  also  as  nitrate  and 
sulphate.  Thus  soil  bacteria  help  to  furnish  shell  fish  and  other  forms 
of  marine  life,  the  material  necessary  for  the  building  of  their  skeletons. 
In  the  course  of  ages  the  latter  become  a  portion  of  the  solid  land  and  as 
coral  reefs,  chalk  cliffs  and  marl  beds  offer  to  microorganisms  a  new 
opportunity  to  start  calcium  carbonate  on  its  migrations. 

EFFECT  OF  CALCIUM  AND  MAGNESIUM  COMPOUNDS  ON  BACTERIAL 
ACTIVITIES.  —  Being  basic  in  character  calcium  and  magnesium  car- 
bonates are  of  great  service  in  maintaining  a  suitable  reaction  in  the 
soil.  But  somewhat  apart  from  this  service  calcium  and  magnesium 
compounds  seem  to  be  particularly  important  for  the  growth  of  certain 
organisms.  It  has  already  been  observed  by  Winogradski  and  Ome- 
lianski  that  magnesium  carbonate  is  especially  useful  in  facilitating  the 


420  MICROBIOLOGY   OF   SOIL 

isolation  and  culture  of  nitrate  bacteria.  Heinze  and  others  have 
noted  the  favorable  action  of  calcium  carbonate  on  the  growth  of 
Azotobacter,  while  the  beneficial  influence  of  calcium  carbonate  and  sul- 
phate on  the  development  of  Ps.  radicicola  has  been  repeatedly  observed 
by  different  investigators. 

Lipman  and  Burgess  found  that  calcium  carbonate  stimulates 
nitrogen  fixation  by  A.  chroococcum  in  solution,  but  is  without  effect 
in  soil.  Magnesium  carbonate  is  very  toxic  both  in  soil  and  in  solution 
for  cultures  of  A.  chroococcum  even  in  concentration  of  o.i  per  cent. 
Calcium  carbonate  exercises  a  protective  action  against  the  toxic 
properties  of  magnesium  carbonate. 

PHOSPHORUS 

AVAILABILITY  OF  PHOSPHATES. — Phosphorus  exists  in  the  soil  largely 
in  the  form  of  phosphates  of  calcium,  magnesium,  iron  and  aluminum. 
A  small  portion  of  it  occurs  in  organic  combination  in  lecithin,  phytin 
and  other  compounds.  The  soil  phosphates  possess  a  very  slight  degree 
of  solubility  and  often  fail  to  become  available  rapidly  enough  to  meet 
the  demands  of  the  growing  crop.  Fortunately  the  presence  of 
carbon  dioxide  generated  from  decaying  organic  matter  hastens  the 
solution  of  the  inert  phosphates,  thus: 

Ca3(P04)2  +  2C02  +  2H20  =  Ca2H2(P04)2  +  Ca(HCO3)2 

For  this  reason  a  maximum  supply  of  available  phosphates  may  be 
secured  by  plants  in  the  presence  of  readily  decomposable  organic 
matter. 

Apart  from  carbon  dioxide  as  a  means  for  making  available  inert 
phosphates,  bacteria  produce  organic  and  inorganic  acids  that  are  of 
direct  service.  The  influence  of  nitrous,  nitric  and  sulphuric  acids,  all 
of  them  products  of  bacterial  activity,  is  undoubtedly  of  some  im- 
portance. The  influence  of  lactic,  acetic  and  butyric  acids,  as  well  as 
of  the  more  complex  humic  acids,  must  be  of  considerable  moment. 
For  instance,  in  the  decomposition  of  bone  meal  by  B.  mycoides, 
Stoklasa  found  that  23  per  cent  of  the  phosphoric  acid  had  become 
soluble,  whereas  in  similar  uninoculated  portions  of  bone  meal  only  3 
per  cent  of  soluble  phosphoric  acid  was  found.  The  significance  of 
organic  acids  produced  by  microorganisms  is.  brought  out  even  more 
strongly  in  the  loss  of  phosphates  from  acid  soils. 


CHANGES    IN   INOEGANIC   CONSTITUENTS  421 

In  so  far  as  the  organic  phosphorus  compounds  are  concerned  bac- 
terial activities  are  important  in  that  the  processes  of  decay  restore  the 
phosphorus  to  circulation.  Hence,  it  will  be  seen  that  microorganisms 
are  directly  concerned  in  the  migration  of  phosphorus  from  the  soil  to 
the  plant  and  from  the  plant  back  to  the  soil. 

RELATION  OF  PHOSPHORUS  TO  DECAY  AND  NITROGEN  FIXATION.— 
Just  as  bacteria  influence  the  transformation  of  phosphorus  compounds 
in  the  soil,  so  phosphorus  itself  affects  the  growth  and  activities  of 
bacteria.  As  one  of  the  essential  constituents  of  living  cells  it  reacts 
on  the  growth  of  microorganisms  and  influences  species  relationships. 
There  are  undoubtedly  species  whose  phosphorus  requirement  is  greater 
than  that  of  other  species.  Indeed,  conditions  may  arise  that  favor  the 
rapid  assimilation  of  soluble  phosphates  by  bacteria.  In  that  case  the 
microorganisms  would  act  as  competitors  to  the  higher  plants.  Among 
the  species  favorably  affected  by  an  abundant  supply  of  phosphates 
Azotobacter  is  quite  prominent.  Hence  nitrogen  fixation  is  in  a  meas- 
ure dependent  upon  a  proper  supply  of  phosphorus  compounds. 

Fred  and  Hart  have  shown  that  the  potassium  ion  does  not  mate- 
rially influence  ammonincation;  soluble  phosphates  cause  large  increases 
in  the  number  of  bacteria,  ammonification  and  carbon  dioxide  produc- 
tion. By  applying  soluble  phosphates  to  the  soil  crop  production  is 
increased,  and  it  is  due,  in  part,  to  the  promotion  of  bacterial  activity. 
The  increased  bacterial  activity  results  in  a  more  rapid  decomposition 
of  the  organic  matter,  thus  making  available  for  the  growth  of  crops 
larger  quantities  of  nitrogen  and  probably  of  minerals. 

SULPHUR 

SULPHUR  COMPOUNDS  IN  THE  SOIL. — Sulphur  occurs  in  the  soil  in 
the  form  of  sulphates  and  in  that  of  organic  compounds.  In  ill- 
aerated  soils  the  reduction  products  of  sulphates,  viz.,  sulphites,  sul- 
phides and  even  elementary  sulphur,  may  be  present  in  small  amounts 
as  a  transition  stage.  According  to  Berthelot  and  Andre  the  protein 
compounds  of  the  soil  humus  are  quantitatively  more  important  than 
the  sulphates.  However,  this  is  not  true  of  arid  and  semi-arid  soils 
in  which  sulphates  represent  a  larger  store  of  combined  sulphur  than 
is  contained  in  organic  substances. 

Sulphur-phosphate  Composts. — In  the  composting  of  sulphur, 
ground  rock  phosphate  (floats)  and  soil  certain  soil  bacteria  oxidize 


422  MICROBIOLOGY    OF    SOIL 

the  sulphur  into  sulphuric  acid,  which  acts  upon  the  insoluble  rock 
phosphate  and  makes  it  soluble  and  available  for  higher  plants.  The 
best  combination  found  at  the  New  Jersey  Agricultural  Experiment 
Station  consists  of  100  parts  of  soil,  120  parts  of  sulphur  and  400  parts 
of  rock  phosphate,  inoculated  with  material  from  an  old  compost. 
The  bacteria  causing  the  oxidation  of  sulphur  were  isolated  at  the  New 
Jersey  Agricultural  Experiment  Station  and  were  found  to  be  short, 
non-motile,  Gram-positive  rods.  They  are  obligate  aerobes  and  are 
able  to  convert  sulphur  into  sulphuric  acid. 

SULPHUR  BACTERIA.  —  In  the  decomposition  of  protein  compounds 
with  a  limited  supply  of  air,  hydrogen  sulphide  and  mercaptans  are 
evolved.  The  quantities  of  hydrogen  sulphide  produced  may  be 
large  enough  to  become  perceptible  to  the  sense  of  smell,  as  happens  in 
the  putrefaction  of  eggs.  At  -the  bottom  of  seas,  rivers,  lakes  and 
ponds  (in  canals,  ditches,  swamps,  etc.)  as  well  as  in  finer-grained  soils 
the  production  of  hydrogen  sulphide  goes  on  almost  uninterruptedly 
owing  to  the  activities  of  a  great  variety  of  bacteria.  The  hydrogen 
sulphide  thus  generated  serves  as  a  source  of  energy  to  a  group  of 
organisms  known  as  sulphur  bacteria.  The  oxidation  of  the  hy- 
drogen sulphide  by  these  bacteria  may  be  expressed  by  the  following 
equations: 

2H2S  +  O2  =  2H2O  +  S2 
S2  +  2O2  =  2SO2 

The  sulphur  dioxide  produced  is  further  changed  into  sulphuric  acid 
in  the  presence  of  oxygen  and  water.  In  its  turn  the  acid  reacts  with 
some  base,  usually  calcium  carbonate,  resulting  in  the  formation  of 
calcium  sulphate.  Thus: 

SO2+O+H2O  =  H2SO4 


We  owe  much  of  our  knowledge  concerning  the  sulphur  bacteria  to 
Winogradski.  This  investigator  showed  that  in  places  where  hydrogen 
sulphide  is  generated  in  considerable  quantities  sulphur  bacteria  grow 
vigorously  and  accumulate  granules  of  sulphur  within  their  cells. 
When  the  cells  containing  sulphur  granules  are  removed  to  suitable 
media,  in  which  no  hydrogen  sulphide  is  present,  the  sulphur  seems 
to  be  gradually  oxidized  and  disappears  and  the  bacteria  finally  die  of 


CHANGES    IN    INORGANIC    CONSTITUENTS  423 

starvation.  Thanks  to  the  sulphur  bacteria,  the  higher  plants  are 
enabled  to  utilize  again  the  sulphur  once  locked  up  in  plant  and  ani- 
mal tissues,  and  liberated  thence  by  decay  bacteria.  The  circulation 
of  sulphur  is  thus  made  possible  and  the  cycle  is  completed  when  the 
sulphates  are  again  used  by  plants  to  build  protein  compounds.  It 
may  also  be  noted  in  this  connection  that  "  Thiobacillus  denitrificans  " 
described  by  Beyerinck,  may  also  oxidize  elementary  sulphur.  In 
this  case,  however,  the  oxygen  is  derived  from  nitrates  instead  of  the 
atmosphere.  Thus: 

6KN03  +  58  +  2CaC03  =  3K2SO4  +  2CaSO4  +  2CO2  +  sN2 

SULPHOFICATION. — Lint  has  found  that  under  optimum  temperature 
and  moisture  conditions,  sulphur  applied  at  the  rate  of  600  pounds 
per  acre  was  almost  completely  oxidized  within  ten  weeks.  Boullanger 
and  Dugardin  in  explaining  the  fertilizing  action  of  sulphur  on  the 
basis  of  its  effect  on  the  supply  of  available  nitrogen  found  that  am- 
monification  was  increased  by  small  amounts  of  sulphur,  nitrogen- 
fixation  was  not  affected  and  nitrification  was  depressed.  It  has  been 
pointed  out  by  Kossovitch,  Brioux  and  Puerbet  that  the  mechanism 
of  sulphur  fertilization  is  very  complex  and  that  the  oxidation  of  free 
sulphur  occurs  entirely  by  bacterial  and  not  by  chemical  means. 
Brown  and  Kellogg  have  recently  advanced  evidence  to  prove  that 
soils  have  a  definite  sulphofying  power  which  is  determinable  in  the 
laboratory  by  a  newly  devised  method.  They  claim  that  the  process 
of  sulphofication  is  mainly  brought  about  by  bacterial  action,  but 
probably  there  is  also  a  small  production  of  sulphates  in  soils  due  to 
chemical  action. 

It  has  been  observed  that  soils  differentiated  by  various  treatments, 
vary  widely  in  sulphofying  power,  the  presence  of  organic  matter  being 
responsible  for  an  increase  up  to  a  certain  point.  Aeration  and  mois- 
ture must  be  optimum  for  favorable  sulphofication  while  the  addition 
of  carbohydrates  to  soils  depresses  the  process. 

SULPHATE  REDUCTION. — The  fact  that  sulphates  may  be  reduced  to 
sulphides  in  the  presence  of  organic  matter  has  been  known  for  many 
years.  In  compost  heaps,  and  at  the  bottom  of  seas,  lakes  and  rivers, 
the  reduction  of  calcium  sulphate  is  of  common  occurrence.  Similarly, 
ferrous  sulphate  may  be  reduced  in  water-logged  soils  and  in  swamps 


424  MICROBIOLOGY   OF    SOIL 

and  may  give  rise  to  deposits  of  bog  iron.  But  while  sulphate  reduction 
is  of  common  occurrence  in  certain  localities,  it  has  been  shown  by  Bey- 
erinck  and  also  by  van  Delden,  that  the  reduction  can  be  accomplished 
in  artificial  media  by  specific  microorganisms.  Two  species  isolated  by 
these  investigators  have  been  named  Sp.  desulphuricans  and  Msp. 
cestuarU.  When  grown  under  anaerobic  conditions  in  culture  media 
supplied  with  combined  nitrogen  and  organic  nutrients  these  organisms 
were  found  capable  of  reducing  sulphates.  The  oxygen  withdrawn 
from  the  sulphates  was  used  for  the  oxidation  of  organic  matter  in  a 
manner  analogous  to  that  in  nitrate  reduction  where  the  oxygen  is 
derived  from  the  nitrates.  Apart  from  the  two  organisms  that  cause 
the  specific  reactions  just  noted,  there  are  many  common  soil  bacteria 
that  may  be  responsible  for  sulphate  reduction  in  a  less  direct  manner. 
Nadson  has  observed  that  when  the  supply  of  oxygen  is  limited  calcium 
sulphate  may  be  reduced  to  sulphide  by  B.  mycoides  and  by  B.  (Proteus) 
vulgaris.  The  calcium  sulphide  according  to  him  may  react  with  car- 
bon dioxide  and  water,  giving  rise  to  the  formation  of  hydrogen  sul- 
phide. Thus: 

CaS  +  CO2  +  H2O  =  CaCO3  +  H2S 

The  hydrogen  sulphide  derived  from  sulphates  or  from  proteins 
becomes  a  source  of  energy  to  the  sulphur  bacteria  as  already  noted  in 
the  preceding  pages. 

POTASSIUM 

THE  TRANSFORMATION  OF  POTASSIUM  COMPOUNDS  IN  THE  SOIL. — 
Potassium  occurs  in  the  soil  largely  in  the  form  of  silicate  minerals. 
Smaller  amounts  occur  as  nitrate,  carbonate  and  in  organic  compounds. 
The  portion  present  as  silicates  is  often  very  large  in  clay-loam  soils, 
amounting  not  infrequently  to  22,679  kg.  to  34,019  kg.  (50,000  to 
75,000  pounds)  per  acre-foot.  Unfortunately  for  the  farmer,  the  grow- 
ing crops  fail,  in  many  cases,  to  secure  sufficient  quantities  of  available 
potash  for  their  rapid  development,  notwithstanding  these  enormous 
stores  of  potassium  compounds.  However,  when  sufficient  quantities 
of  readily  fermentable  organic  matter  are  present  and  the  generation  of 
carbon  dioxide  is  rapid  the  silicates  weather  sufficiently  fast  to  meet 
the  demands  of  maximum  harvests.  The  part  played  by  carbon  dioxide 


CHANGES   IN   INORGANIC   CONSTITUENTS  425 

in  the  transformation  of  inert  potash  compounds  may  be  illustrated  by 
the  following  reaction: 


A12O3K2O  6SiO2  +  CO2  +  2H2O  =  A12O3  2Si02  2H2O  +  K2CO3 

Under  actual  conditions  it  is  the  aim  of  the  farmer  to  stimulate 
bacteria]  activities  (and,  therefore,  the  production  of  carbon  dioxide)  in 
his  land  by  the  use  of  animal  manures  or  green  manures  and  of  com- 
mercial fertilizers.  Apart  from  the  influence  of  carbon  dioxide  avail- 
able potash  compounds  may  likewise  be  formed  on  account  of  nitric, 
sulphuric,  acetic,  lactic,  butyric  and  other  acids  produced  by  different 
soil  bacteria. 

OTHER  MINERAL  CONSTITUENTS 

IRON.  —  The  investigations  of  Ehrenberg,  Winogradski,  Molisch, 
Adler,  Ellis  and  others  have  accumulated  a  mass  of  data  relating  to  the 
so-called  iron  bacteria.  These  organisms  belong  to  the  class  of  higher 
bacteria  and  recently  forms,  such  as  rod-shaped  bacteria,  have  been 
isolated  which  have  a  marked  ability  to  precipitate  iron  oxide  out  of 
solutions  of  iron  salts.  Winogradski  believed  that  the  reaction  is  a 
physiological  one  in  that  the  microorganisms  oxidize  ferrous  to  ferric 
compounds,  and  utilize  for  their  growth  the  energy  thus  made  available. 
The  investigations  of  Molisch,  Adler  and  Ellis  show,  however,  that  the 
iron  bacteria  can  exist  very  well  without  iron  compounds  and  that  the 
precipitation  of  iron  oxide  is  due  to  mechanical  rather  than  chemical 
influences.  But  whether  physiological  or  mechanical  the  influence  of 
these  microorganisms  is  felt  in  the  formation  of  bog  iron,  and  in  the 
filling  up  of  iron  pipes;  in  the  latter  instance  much  annoyance  is  occa- 
sionally experienced  by  those  in  charge  of  municipal  water  supplies. 

Compounds  of  iron  are  of  considerable  significance  in  the  life 
processes  of  many  bacterial  species.  For  instance,  it  was  shown  by 
Lipman  and  after  him  by  Koch,  that  Azotobacter  will  not  develop  in  cul- 
ture media  devoid  of  iron  compounds.  In  field  practice  small  applica- 
tions of  ferrous  sulphate  often  seem  to  exert  a  favorable  effect  on  crop 
growth,  and  there  is  reason  to  suspect  that  soil-microbial  activities  are 
of  some  moment  in  bringing  about  the  results  noted. 

ALUMINUM,  MANGANESE,  COPPER.  —  Weathering  processes  and  the 
relation  of  carbon  dioxide  to  these  processes  have  already  been  dis- 
cussed in  connection  with  calcium  and  potassium  compounds.  To  a 


426  MICROBIOLOGY   OF    SOIL 

great  extent  aluminum  is  affected  by  these  reactions,  for  in  the  decompo- 
sition of  feldspar,  kaolinite  is  one  of  the  important  products  formed. 
Hence,  bacteria  become  a  factor  of  considerable  importance  in  the  forma- 
tion of  hydrated  silicates  of  aluminum,  at  least,  in  the  presence  of 
organic  matter.  Moreover,  it  is  recognized  in  the  ceramic  industries 
that  after  it  is  dug  clay  must  undergo  ripening  in  order  to  be  suitable 
for  certain  purposes.  The  ripening  process  involves  the  activities  of 
bacteria.  Unfortunately  very  little  is  known  about  the  reactions  that 
occur  in  the  ripening  of  clay. 

As  to  manganese  and  copper  there  is  scarcely  any  experimental  evi- 
dence available  as  to  the  part  played  by  their  compounds  in  the  soil, 
particularly  in  so  far  as  they  affect  microorganic  life.  To  some  extent, 
it  is  known  that  where  Bordeaux  mixture  has  been  employed  for  spray- 
ing potatoes,  cranberries,  fruit  trees,  etc.,  plant  growth  is  subsequently 
stimulated  to  a  striking  extent.  In  view  of  the  very  slight  quantities  of 
copper  that  are  actually  added  to  the  soil  by  these  sprays,  it  is  possible 
that  the  effects  noted  are  caused  by  stimulated  or  changed  microbial 
activities.  This  view  finds  some  support  in  the  influence  exerted  by 
copper  sulphate  on  the  growth  of  algae  in  lakes,  ponds,  and  shallow 
streams. 

It  has  also  been  reported  that  the  decomposition  of  complex  silicates 
has  been  effected  from  powdered  minerals  by  nitrite  bacteria. 

ANTAGONISM 

A  subject  which  bids  fair  to  become  a  fertile  source  of  investigation 
is  the  application  of  certain  biochemical  laws,  as  established  by  Loeb 
and  Osterhout  in  the  animal  and  plant  worlds  respectively,  to  the 
effect  of  salts  on  the  physiological  efficiency  of  soil  bacteria  in  pure  and 
mixed  cultures,  as  well  as  in  the  soil.  C.  B.  Lipman  has  advanced  in- 
formation concerning  the  antagonism  between  anions  as  related  to 
nitrogen  transformations  in  soils,  with  special  reference  to  the  reclama- 
tion of  alkali  lands.  Antagonism  exists  to  a  more  or  less  marked 
extent  between  anions  of  alkali  salts  (as  for  example  between  NaCl 
and  Na2SO4,  Na2CO3  and  Na2SO4  and  between  NaCl  and  Na2CO3) 
when  the  ammonifying  or  nitrifying  powers  of  the  soil  are  employed 
as  criteria.  The  nitrogen-fixing  flora,  however,  is  not  similarly 
affected,  apparently  offering  greater  resistance.  The  practical  sug- 


CHANGES   IN    INORGANIC    CONSTITUENTS  427 

gestion  carried  out  of  such  data  then,  involves  the  addition  of  salts 
to  the  toxic  salts  already  contained  in  a  given  soil,  and  thereby  im- 
proving its  ammonifying  and  nitrifying  power. 

VARIABILITY  IN  SOIL  FERTILITY  INVESTIGATIONS 

Waynick  has  pointed  out  that  the  variations  between  different  soil 
samples  taken  from  a  small  area  may  be  of  such  magnitudes  as  to  throw 
doubt  upon  the  validity  of  the  experimental  data  obtained  with  one  or 
a  limited  number  of  samples.  A  single  sample  of  any  soil  is  of  little 
value  as  regards  determinations  which  may  be  made  upon  it.  A  com- 
posite may  be  considered  of  value  only  after  the  probable  error  to  which 
it  is  subject  is  known  and  this  can  only  be  determined  by  the  use 
of  a  large  number  of  individual  samples. 


DIVISION  IV 

MICROBIOLOGY  OF  MILK  AND  MILK  PRODUCTS 


CHAPTER  I* 

THE  RELATION  OF  MICROORGANISMS  TO  MILK 

CHARACTER  OP  MILK 

The  ideal  milk  is  that  which  reaches  the  consumer  in  as  nearly  as 
possible  the  condition  in  which  it  leaves  the  udder  of  the  healthy  cow. 

The  factors  which  determine  the  quality  of  commercial  milk  may  be 
stated  as  follows:  (a)  Food  value,  (b)  flavor  and  odor,  (c)  keeping 
quality,  (d)  cleanliness,  (e)  healthfulness.  With  the  exception  of  the 
first,  all  of  these  qualities  are  in  part  or  wholly  dependent  upon  the 
microbial  content  of  the  milk. 

Fresh  normal  milk  has  a  pleasant  taste  and  aroma  and  is  gener-. 
ally  liked  as  a  food  or  drink;  but  unless  properly  cared  for  will  not 
long  remain  in  its  normal  condition.  No  article  of  human  diet  is 
more  susceptible  to  undesirable  changes,  due  to  the  delicate  nature 
of  the  milk  itself  and  to  the  conditions  naturally  surrounding  its  pro- 
duction and  handling.  The  injurious  changes  which  commonly  occur 
in  milk  are  of  two  kinds. 

ABSORBED  TAINTS  AND  ODORS 

Milk  is  very  quickly  affected  by  odors  of  any  sort.  The  foreign 
odor  may  be  absorbed  before  the  milk  leaves  the  udder  if  the  cow  has 
eaten  strong  feeds,  such  as  cabbage,  onions,  etc.,  or  it  may  be  absorbed 
after  the  milk  is  drawn  from  the  cow.  If  milk  is  exposed  to  any 

*  Prepared  by  W.  A.  Stocking  with  the  exception  of  the  paragraphs  treating  the  acid-forming 
bacteria,  prepared  by  E.  G.  Hastings. 

428 


THE   RELATION   OF   MICROORGANISMS   TO   MILK  429 

strong  odor,  such  as  silage  or  foul  air,  resulting  from  lack  of  ventila- 
tion in  the  stable  at  milking  time,  these  odors  will  be  taken  up  by  the 
milk  with  surprising  rapidity.  If  placed  in  an  ice  chest  with  fresh 
strawberries  or  pineapple,  or  foods  like  cabbage  or  turnips,  the  milk 
will  very  quickly  absorb  the  odor  of  these  foods.  The  absorption 
of  any  foreign  odor  gives  to  milk  a  decidedly  disagreeable  taste.  This 
is  true  even  when  the  odor  which  is  absorbed  is  pleasant  in  itself  as 
in  the  case  of  strawberries  or  pineapples.  When  the  "off"  flavors  are 
due  to  absorption  they  are  strongest  at  the  outset  and  become  less 
pronounced  as  the  milk  becomes  older,  especially  if  it  is  subjected  to 
some  method  of  aeration. 

CHANGES  DUE  TO  MICROORGANISMS 

While  absorption  of  foreign  odors  is  not  uncommon,  probably 
most  of  the  undesirable  flavors,  found  in  milk  when  it  reaches  the 
consumer,  are  caused  not  by  absorption  but  by  the  growth  of 
microorganisms  in  the  milk.  In  this  class  the  changes  are  slight  at 
first  and  increase  with  the  age  of  the  milk.  Changes  of  this  sort 
include  the  common  phenomena  of  souring  and  curdling,  the  so- 
called  sweet  curdling,  ropy  or  slimy  milk,  bitter  flavors,  gassy  milk 
and  a  large  variety  of  changes  usually  known  as  barny  or  cowy  odors 
and  flavors.  If  milk  could  be  kept  free  from  microorganisms,  it 
might  be  kept  for  some  time  without  showing  perceptible  changes  in 
appearance  or  taste.  No  other  food  product  will  undergo  fermenta- 
tion changes  as  rapidly  as  milk  because  it  is  an  ideal  culture  medium 
for  the  growth  of  most  kinds  of  microorganisms,  especially  bacteria 
and  yeasts.  Not  only  does  milk  contain  the  needed  food  elements  but, 
being  in  liquid  form,  they  are  easily  available  for  the  use  of  micro- 
organisms. The  proteins  and  milk  sugar  are  most  easily  attacked 
and  it  is  the  breaking  down  of  these  which  causes  most  of  the  changes 
in  the  milk. 

MICROBIAL  CONTENT  or  MILK 

The  amount  of  care  exercised  in  the  production  and  handling  is 
a  most  important  factor  in  determining  the  bacterial  contamina- 
tion of  milk.  On  this  basis  milk  may  be  roughly  divided  into  three 
classes. 


430 


MICROBIOLOGY   OF   MILK    AND    MILK   PRODUCTS 


COMMON  MILK. — When  we  recognize  the  extreme  ease  with  which 
milk  undergoes  bacterial  changes,  we  are  not  surprised  to  find  that 
ordinary  milk,  when  delivered  to  the  consumer,  contains  relatively 
large  numbers  of  bacteria.  Age  is  one  of  the  chief  factors  in  de- 
termining the  germ  content  of  milk.  We,  therefore,  expect  to  find 
the  milk  in  large  cities  having  a  much  higher  germ  content  than  in 
smaller  cities  and  towns.  The  normal  germ  content  of  ordinary 
milk  as  it  is  found  in  the  cities  may  be  shown  by  the  following 
tables. 

BACTERIA   IN   BOSTON   MILK* 

Average  taken  from  2,394  Samples 
From  June  to  September 

Per  cent 

Below  100,000  bacteria  per  c.c 42 .  o 

Between  100,000  and  500,000  per  c.c 29. 75 

Between  500,000  and  1,000,000  per  c.c 9.75 

Between  1,000,000  and  5,000,000  per  c.c 12 . 75 

Above  5,000,000  per  c.c 5.0 

Uncountable  plates 0.75 

BACTERIAL  COUNTS  OF  CHICAGO  (RAW)  MILK| 

Date  Number  of  Average  Lowest  Highest 

samples  count  count  count 

January |  64  1,067,000  27,000  5,500,000 

April 43  5,948,000  14,000  150,000,000 

July 183  12,548,000  8,000  190,000,000 

BACTERIA  IN  MILK  OF  CONNECTICUT  CITIES! 

Bacterial  count  Number  of  samples 

Under  50,000 1,707 

50,000-100,000 , 130* 

100,000-500,000 459 

500,000-1,000,000 98 

Over  i ,000,000 73 

These  figures  give  the  results  of  2,467  samples  collected  in  seventy-five  different 
towns  in  the  State  covering  a  period  of  one  entire  year. 

Goler  gives  the  average  bacterial  count  for  1,057  samples  of  market  milk  collected 
in  Rochester  during  the  year  1909  as  446,099  per  c.c.  Of  these  samples  1.79  per  cent 
were  above  5,000,000  and  38.4  per  cent  below  100,000. 

*  Data  given  by  Hill  and  Slack, 
t  Data  given  by-'Tonney. 
t  Data  given  by  Conn. 


THE   RELATION   OF   MICROORGANISMS    TO   MILK  431 

In  Montclair,  N.  J.,  the  average  bacterial  count  for  the  year  1918, 
for  the  fifteen  producers  who  delivered  raw  milk,  was  as  follows: 


BACTERIAL  COUNTS  OF  RAW  MILK,  MONTCLAIR,  N.  J.,  1918 

Producer's  X<>.  Average  Count 

1  6,OOO 

2  10,500 

3  •  20,000 

4  37,000 

5  45,3oo 

6  47,000 

7  53,ooo 

8  65,500 

9  68,000 

10  7S,ooo 

11  82,000 

12  82,000 

13  90,000 

14  171,000 

15  226,000 
Average  71,886 


In  Ithaca,  N.  Y.,  samples  taken  for  the  year  1919  gave  average 
bacterial  counts  by  months  as  follows: 

BACTERIAL  COUNTS  OF  MILK  IN  ITHACA,  N.  Y.,  1919 

Month  Average  Count 

January 111,450 

February . ' 145,990 

March .' 101,050 

April 93,46o 

May 123,320 

June 115,865 

July '.  66,525 

August 47,620 

September 151,260 

October 11,030 

November 27,120 

December 91,700 


432  MICROBIOLOGY    OF    MILK   AND  .MILK   PRODUCTS 

The  immense  numbers  of  bacteria  found  in  milk  in  the  large  cities 
are  usually  the  result  of  the  rapid  growth  of  the  Bad.  lactis  acidi  group 
resulting  from  the  age  of  the  milk  and  the  temperature  at  which  it  has 
been  kept.  Such  milk  may  also  contain  large  numbers  of  those  sapro- 
phytic  organisms  which  occur  freely  in  nature  and  which  may  be 
abundant  about  the  stables  and  milk-house.  The  number  of  this  group 
depends  largely  upon  the  sanitary  conditions  of  production  and  the 
initial  contamination.  In  ordinary  milk  organisms  of  the  Bact.  lactis 
acidi  type  will  constitute  a  very  large  percentage  of  those  present  when 
the  milk  reaches  the  city  even  before  it  shows  any  perceptible  signs  of 
souring.  During  the  past  few  years  great  progress  has  been  made  in  the 
production  of  clean  milk  and  at  present  quite  an  important  part  of  the 
general  raw  milk  supply  of  our  cities  has  a  very  much  lower  germ  con- 
tent than  it  had  a  few  years  ago. 

SPECIAL  MILKS. — In  this  class  may  be  considered  those  milks  known 
as  Selected,  Inspected,  or  Guaranteed.  As  commonly  used  these  terms 
mean  milk  which  has  been  produced  and  handled  with  considerably 
more  care  than  ordinary  market  milk  but  not  with  the  extreme  care 
required  for  certified  milk.  While  these  and  similar  terms  do  not  always 
mean  milk  of  the  same  grade  in  different  places,  they  usually  mean  milk 
produced  by  herds  which  have  been  shown  by  the  tuberculin  test  to  be 
free  from  tuberculosis.  Considerable  care  is  exercised  in  all  the  opera- 
tions of  handling  the  milk.  The  result  is  that  these  milks  usually  have 
a  much  lower  germ  content  than  the  ordinary  milk  supply  of  the  same 
city.  Sometimes  the  germ  content  of  such  milk  compares  favorably 
with  that  of  certified  milk.  These  milks  may  contain  various  types  of 
normal  milk  organisms  but  they  should  not  contain  any  tubercle 
bacteria. 

CERTIFIED  MILK. — Certified  milk  means  milk  which  has  been  pro- 
duced according  to  the  regulations  of  and  under  the  supervision  of  a 
medical  milk  commission.  The  stables  and  cows  are  kept  extremely 
clean.  No  dust  is  allowed  in  the  stable  at  milking  time.  The  cow's 
flanks  and  udder  are  washed  just  before  milking,  the  milkers  wear  white 
suits  and  wash  their  hands  before  milking  each  cow.  Small-top  pails 
are  used  and  the  milk  is  cooled  as  soon  as  drawn  from  the  cow.  The 
extreme  care  exercised  in  the  production  and  handling  of  this  milk  has 
a  very  marked  effect  on  the  number  of  bacteria  found  in  it.  The  follow- 
ing counts  are  typical  of  certified  milk. 


THE   RELATION   OF   MICROORGANISMS    TO   MILK 


433 


BACTERIAL  COUNTS  OF  CERTIFIED  MILK  IN  DIFFERENT  CITIES 
Boston,  Oct.  i,  1909  to  Sept.  30,  1910* 


Farm  number 

Number  samples 

Average  bacteria  count 

I 

17 

5,794 

2 

13 

4,176 

3 

30 

6,825 

4 

12 

i,47S 

5 

7 

2,294 

New  York  City,  Oct.,  1909  to  Sept. 


Farm  number 

Average  bacteria  count 

I 

11,132 

2 

10,516 

3 

8,504 

4 

16,193 

5 

2,863 

6 

11,246 

7 

23,705 

8 

5,370 

9 

15,062 

10 

459 

Chicago  t 

Farm  number 

Number  samples                   Average  bacteria  count 

I 

5i                                           5,6i2 

2 

60                                          4,078 

3 

43                                           6,502 

4 

17                                        2,553 

Brooklyn 

Moak  gives  the  average  of  321  counts  for  certified  milk  delivered  in  Brooklyn 
during  the  first  six  months  of  1910  as  4,095  bacteria  per  c.c.  The  best  average  from 
any  one  farm  was  561  bacteria  per  c.c. 

*  Data  given  by  Arms, 
t  Data  given  by  Park. 
J  Data  by  Heinemann. 
28 


434  MICROBIOLOGY   OF   MILK   AND   MILK   PRODUCTS 

SOURCES  OF  MICROORGANISMS  IN  MILK 

The  sources  from  which  bacteria  get  into  the  milk  have  been  the  sub- 
ject of  much  investigation  during  the  past  few  years,  until  now  the  chief 
sources  of  contamination  are  pretty  well  understood.  These  sources 
may  be  grouped  in  a  general  way  under  the  following  heads: 


FIG.  138. — Vertical  section  of  one  quarter  of  udder  showing  teat,  milk  cistern,  and 
larger  milk  ducts.     (After  Ward  and  Hopkins.} 

INTERIOR  OF  THE  Cow's  UDDER.  Healthy  Udders. — Milk  as  it  is 
secreted  by  the  normal  udder  of  a  healthy  cow  is  probably  free  from 
bacteria.  It  is  very  difficult,  however,  to  obtain  milk  from  the  udder 


THE    RELATION    OF    MICROORGANISMS    TO    MILK  435 

which  does  not  contain  bacteria  in  greater  or  less  numbers.  This  is  due 
to  the  fact  that  immediately  after  secretion  the  milk  becomes  contami- 
nated by  bacteria  which  exist  in  the  interior  of  the  udder.  Early  inves- 
tigators, notably  de  Freudenreich  and  Grotenfelt,  believed  that  milk 
while  in  the  udder  was  entirely  free  from  microorganisms.  Later  inves- 
tigations, however,  by  Moore,  Ward,  Bolley,  Hall  and  others,  have 
shown  that  the  healthy  udder  normally  contains  bacteria  in  appreciable 
numbers.  It  has  been  found  that  bacteria  are  present  even  in  the  upper 
portions  of  the  udder  in  the  small  milk  passages  leading  from  the  se- 
creting cells.  These  organisms,  which  normally  exist  in  the  milk  pas- 
sages of  the  udder,  gain  entrance  through  the  orifice  in  the  end  of  the 
teat  where  they  find  suitable  conditions  for  growth  and,  once  inside, 
work  up  through  the  milk  cistern  to  the  larger  milk  ducts  and  finally 
though  all  parts  of  the  udder  (Fig.  138).  The  number  of  bacteria  found 
in  the  udder  varies  widely  in  different  cows  as  may  be  seen  by  the 
following  figures: 

BACTERIAL  CONTENT  OF  ENTIRE  MILK  OF  DIFFERENT  Cows 

Cow  No.  i 850  bacteria  per  c.c. 

Cow  No.  2 750  bacteria  per  c.c. 

Cow  No.  3 25  bacteria  per  c.c. 

Cow  No.  4 112  bacteria  per  c.c. 

Cow  No.  5 70  bacteria  per  c.c. 

Cow  No.  6 1*850  bacteria  per  c.c. 

If  portions  of  milk  are  taken  at  different  intervals  during  the  process 
of  milking  in  such  a  way  that  all  external  contamination  is  prevented,  it 
will  be  found  that  the  first  few  streams  of  " fore-milk"  contain  many 
more  organisms  than  the  milk  drawn  later.  After  the  first  ten  or  twelve 
streams  the  number  of  organisms  will  decrease  quite  rapidly,  normally 
becoming  less  and  less  until  the  final  strippings,  when  there  is  usually  a 
marked  increase.  This  condition  indicates  that  the  larger  number  of 
organisms  exist  in  the  milk  cistern  and  larger  milk  ducts  in  the  lower 
part  of  the  udder  and  are  therefore  removed  during  the  early  part  of  the 
milking.  The  increase  at  the  end  of  the  milking  is  probably  due  to  the 
greater  manipulation,  resulting  in  dislodging  some  of  the  organisms 
which  have  adhered  to  the  walls  of  the  milk  passages. 

Not  only  does  the  number  of  organisms  in  different  cows  vary,  but 
there  is  a  marked  difference  in  the  different  quarters  of  the  same  udder, 
as  shown  by  the  following  figures. 


436  MICROBIOLOGY   OF   MILK   AND    MILK    PRODUCTS 

BACTERIA  IN  DIFFERENT  QUARTERS  or  Cow's  UDDER* 


Right  front 
quarter  .of 
udder 

Left  front 
quarter  of 
udder 

Right  back 
quarter  of 
udder 

Left  back 
quarter  of 
udder 

No. 
sam- 
ples 

Aver- 
age per 
c.c. 

No. 
sam- 
pies 

Aver- 
age per 
c.c. 

No. 
sam- 
ples 

Aver- 
age per 
c.c. 

No. 
sam- 
ples 

Aver- 
age per 
c.c. 

Herd  of  1900—02.  .  . 

79 
i8S 
46 

419 
199 
161 

249 

77 

174 
46 

378 
130 
107 

191 

80 
i85 
46 

653 
636 

597 
635 

80 
186 
46 

617 
698 

342 

625 

Herd  of  1910—11  

Herd  of  A.  G.  L  
Averages  

Average  germ  content  per  c.c.  in     316  samples  from  herd  of  1900-02 518 

Average  germ  content  per  c.c.  in     730  samples  from  herd  of  1910-11 420 

Average  germ  content  per  c.c.  in     184  samples  from  herd  of  A.  G.  L 320 

Average  germ  content  per  c.c.  in  1,230  samples  from  78  cows 428 

The  number  of  organisms  normally  found  in  the  udder  is  much 
smaller  than  would  be  expected  when  we  consider  the  fact  that  ideal 
conditions  of  food  and  temperature  are  provided  there  for  bacterial 
growth.  The  relatively  small  number  of  organisms  is  perhaps  due  to 
some  germicidal  action  existing  in  the  udder.  Attempts  to  increase  the 
germ  content  in  the  udder  by  injecting  cultures  of  different  species  of 
saprophytic  bacteria  have  failed  to  produce  a  continued  increase,  the 
injected  organisms  usually  decreasing  very  rapidly  in  numbers  until 
they  disappear  at  the  end  of  a  few  days.  From  the  standpoint  of 
ordinary  market  milk,  the  number  of  bacteria  found  in  the  healthy 
udder  is  so  small  that  it  is  of  little  commercial  importance.  In  dairies 
where  a  very  small  germ  content  is  desired,  however,  this  source  of  in- 
fection must  be  taken  into  account  and  in  certain  cases  individual  cows, 
which  normally  have  a  high  bacteria  content  in  the  udder,  can  be  dis- 
carded to  advantage. 

It  is  evident  that  ma'ny  species  do  not  find  the  conditions  in  the 
udder  suitable  for  their  growth,  since  investigations  have  shown  that 
comparatively  few  species  exist  for  any  length  of  time  in  the  healthy 
udder.  Certain  types  of  micrococci  are  the  predominating  forms  with 
occasional  cultures  of  other  species.  The  Bad.  lactis  acidi  type  does 

*  Harding  and  Wilson:     Technical  Bui.  No.  27,  N.  Y.  Agric.  Exp.  Sta.,    1913. 


THE   RELATION   OF   MICROORGANISMS   TO   MILK  437 

not  thrive  in  the  udder.  The  types  of  organisms  commonly  found 
there  do  not  seem  to  develop  rapidly  in  the  milk  when  it  is  held  at  low 
temperatures  and  fail  to  produce  any  appreciable  changes  in  it  during 
the  normal  life  of  market  milk. 


FIG.  139. — Colonies  developing  in  agar  plate  held  for  ten  seconds  in  position  of 
milk  pail  after  udder  was  brushed  gently  with  the  hand. 

Diseased  Udders. — If,  however,  the  cow  is  suffering  from  disease 
in  the  udder,  the  bacterial  condition  may  be  quite  different  from  that 
described  above.  In  this  case,  the  milk  may  be  filled  with  the  specific 
bacteria  before  it  leaves  the  udder.  In  cases  of  inflammatory  trouble  or 
tuberculosis  in  the  udder  the  milk  may  contain  very  large  numbers  of  or- 
ganisms, frequently  many  millions  per  c.c.  at  the  time  the  milk  is  drawn. 


438  MICROBIOLOGY    OF    MILK   AND    MILK   PRODUCTS 

EXTERIOR  OF  Cow's  BODY. — The  nature  of  the  cow's  coat  and 
the  condition  under  which  she  is  normally  kept  favor  the  accumulation 
of  dust  and  bacteria  upon  her  body.  Unless  special  care  is  taken  to 
keep  the  cow's  body  free  from  dirt,  the  organisms  which  fall  into  the 
milk  from  this  source  at  milking  time  will  constitute  one  of  the  most 
important  sources  of  contamination.  The  importance  of  this  source 


FIG.  140. — rColonies  developing  from  cow-hairs  planted  in  agar  plate. 

of  contamination  may  be  recognized  when  we  see  what  large  numbers 
of  microorganisms  may  be  carried  by  small  particles  of  dust  or  an 
individual  cow  hair.  The  amount  of  this  source  of  contamination  is 
indicated  by  the  marked  reduction  in  germ  content  resulting  from  the 
use  of  a  small  top  pail  (page  442). 

.  The  importance  of  this  source  of  contamination  depends  very 
largely  upon  the  conditions  under  which  the  cows  are  kept  and  the  care 
exercised  in  cleaning  just  previous  to  milking.  In  many  of  the  certified 
milk  dairies  this  source  of  contamination  is  reduced  to  a  minimum  and 
has  little  effect  upon  the  milk. 


THE    RELATION    OF   MICROORGANISMS    TO   MILK 


439 


ATMOSPHERE  OF  STABLE  AND  MILK  HOUSE. — The  atmosphere 
of  the  stable  may  be  an  important  factor  in  influencing  the  bacterial 
content  of  fresh  milk  In  well  kept  stables  fairly  free  from  dust  this 
source  of  contamination  is  usually  not  important  but  in  stables  where 
the  air  is  full  of  dust  at  time  of  milking,  the  germ  content  of  the  milk 
may  be  appreciably  increased  from  this  source.  In  sanitary  dairies 
this  factor  is  fully  recognized  and  every  effort  is  made  to  prevent  the 
presence  of  dust  in  the  atmosphere  at  the  time  of  milking. 


FIG.  141. — Colonies  developed  from  a  bit  of  dust  found  in  cow  stable. 

culture. 


Agar  plate 


THE  MILKER. — Not  infrequently  the  milker  himself  is  a  source  of 
contamination.  If  his  clothing  and  hands  are  dirty  or  if  he  brushes 
against  the  cow,  the  dust  thus  dislodged  may  carry  into  the  milk 
large  numbers  of  microorganisms.  This  is  shown  in  the  difference  in 
the  germ  content  of  milk  drawn  by  two  men  milking  in  the  same  barn 
under  identical  conditions. 


440  MICROBIOLOGY   OF   MILK   AND -MILK   PRODUCTS 

DIFFERENCE  IN  NUMBER  OF  BACTERIA  IN  MILK  DRAWN  BY  MEN  IN  SAME  STABLE 


• 

Number  of  milkings 

Number  of  bacteria 
per  c.c. 

Milker  No  i. 

TQ 

2  A^O 

Milker  No.  2  

10 

17  TQO 

THE  UTENSILS. — If  properly  cared  for,  the  dairy  utensils  should 
not  add  to  the  germ  content  of  the  milk.  Not  infrequently,  however, 
they  are  faulty  in  construction.  In  open  seams  and  other  places  the 
milk  may  accumulate  and  not  be  thoroughly  washed  out.  Usually 
when  utensils  of  this  sort  are  used,  the  methods  for  washing  and  ster- 
ilizing are  not  sufficient  and  bacteria  multiply  in  large  numbers  in  the 
cracks  and  crevices  and  contaminate  each  new  lot  of  milk  put  into 
them.  Sometimes  the  utensils  which  are  properly  constructed  may 
contaminate  the  milk  because  they  have  not  been  properly  cleansed 
and  sterilized.  The  possible  effect  of  the  utensils  on  the  germ  content 
of  the  milk  put  into  them  is  shown  by  recent  work  done  at  the  Illinois 
Agricultural  Experiment  Station.*  It  was  found  that  when  the  uten- 
sils were  properly  washed  and  thoroughly  steamed  and  dried  they  did 
not  add  many  bacteria  to  the  milk.  On  the  other  hand,  when  they 
were  not  well  steamed  and  especially  when  allowed  to  stand  wet  for 
several  hours  they  added  very  large  numbers  of  bacteria  to  the  milk. 
This  is  shown  by  the  following  table. 

AVERAGE  NUMBER  OF  BACTERIA  ADDED  TO  FIFTY  LITERS  OF  MILK  BY  THE  VARIOUS 
UNSTEAMED  UTENSILS  IN  WHICH  IT  WAS  HANDLED 


Source  of  bacteria 

Number  of  bacteria 
per  cc.  of  milk 

Total  number 
of  bacteria 

Sources  other  than  utensils 

c.ooo 

250  ooo  ooo 

2  pails 

eJ4,6'?1? 

2,  73I,7<O,OOO 

i  strainer 

7,31"? 

36^  7  <\O  OOO 

i  clarifier  tank  ...                 

8,038 

4OI,9OO,OOO 

i  clarifier 

14.1,340 

7  067  OOO  OOO 

i  cooler     

<\O,QOO 

2,  <4"?,  OOO,  OOO 

i  bottle-filler  tank 

83,246 

4.162  300  ooo 

Total                                    .           

3^0,000 

17  ^23  7OO  OOO 

Total  for  utensils  

34^,000 

17*273,700,000 

*  Illinois  Bull.  204,  1918. 


THE    RELATION    OF   MICROORGANISMS    TO   MILK  441 

These  figures  indicate  that  the  utensils  may  play  a  much  more  impor- 
tant part  in  determining  the  total  germ  content  of  milk  than  was 
formerly  supposed.  The  use  of  steam  is  the  most  efficient  means  of 
sterilizing  all  dairy  utensils,  but  boiling  water  may  give  very  satisfac- 
tory results  if  used  at  actual  boiling  temperature.  If  not  used  at  the 
boiling  temperature  some  of  the  resistant  organisms  will  not  be 
killed  and  will  be  left  to  inoculate  the  fresh  milk.  The  ropy  milk 
organism,  B.  lactis  viscosus,  often  remains  in  the  utensils  from  day  to 
day  in  this  way. 

WATER  SUPPLY. — Sometimes  the  water  used  for  washing  the  dairy 
utensils  is  a  serious  source  of  contamination.  Serious  epidemics  of 
disease  have  been  traced  to  this  source  where  the  utensils  were  washed 
with  water  contaminated  by  typhoid  or  other  disease  organisms 
and  were  not  sufficiently  sterilized  to  kill  those  remaining  in  the  uten- 
sils. Such  dairy  troubles  as  ropy  milk  and  gassy  milk  may  be  caused 
by  the  water  used  for  washing  purposes. 


METHODS  OF  PREVENTING  CONTAMINATION  OF  MILK 

INDIVIDUAL  Cows. — Normally  the  number  of  microorganisms 
found  in  the  udder  is  not  sufficient  to  be  a  serious  source  of  contami- 
nation for  market  milk.  There  are,  however,  certain  cows  which 
have  a  much  higher  germ  content  than  others,  and  where  a  very  low 
count  is  desired  in  the  milk,  it  may  sometimes  be  advisable  to  elimi- 
nate such  cows  from  the  herd. 

CARE  OF  THE  Cow's  BODY. — In  order  to  reduce  to  the  minimum  the 
contamination  from  the  cow's  body,  she  should  be  kept  as  clean  as 
possible.  Dust  should  not  be  allowed  to  accumulate  in  her  coat. 
It  is  well  to  keep  the  hair  of  the  flank  and  udder  clipped  in  order  to 
prevent  the  accumulation  of  dust  and  also  to  facilitate  the  process  of 
cleaning.  The  use  of  a  damp  cloth  for  wiping  the  flank  and  udder 
at  milking  time  is  a  very  efficient  means  of  reducing  this  source  of 
contamination..  The  beneficial  effect  of  this  method  may  be  seen 
in  the  following  table. 

Even  when  considerable  care  is  taken  to  clean  the  surface  of  the 
cow's  body,  there  will  still  be  some  organisms  which  may  fall  into  the 
pail  at  milking  time.  This  number  can  be  very  materially  lessened 


442 


MICROBIOLOGY   OF   MILK   AND    MILK   PRODUCTS 


EFFECT  OF  WIPING  UDDER  AND  FLANK  WITH  A  DAMP  CLOTH  AS  SHOWN  BY  BACTERIAL 

COUNTS  OF  MILK 


Number  of  experiments 

Date 

Treatment 

Bacteria  per  c.c. 

I 

Apr.  13 

I 
Not  wiped 

2,780 

2 

Apr.  i  < 

Wiped 
Not  wiped 

530 

I;3IO 

Apr.  1  6 

Wiped 
Not  wiped 

3IO 
800 

4... 

May  28 

1               Wiped 
Not  wiped 

754 
1,130 

Wiped 

590 

by  reducing  as  far  as  possible  the  area  through  which  dust  can  fall  into 
the  milk  pail.  This  can  be  accomplished  by  the  use  of  a  milking 
pail  with  a  small  top. 

VALUE  OF  SMALL  TOP  PAIL  IN  REDUCING  GERM  CONTENT  OF  MILK 


Experiment 

Kind  of  pail 

Bacteria  per  c.c.  of  milk 

No.  I                                          .     .  : 

Open 

15,500 

No  2                              ... 

Small  top 
Open 

7,750 
3,700 

No   3 

Small  top 
Open 

1,100 
30,000 

Small  top 

4,700 

12  34 

FIG.  142. — Some  different  styles  of  small  top  milking  pails  which 
are  practical  and  efficient. 


THE    RELATION    OF   MICROORGANISMS    TO   MILK  443 

Results  of  extended  trials  in  different  barns  demonstrate  the  fact 
that  approximately  two- thirds  of  the  organisms  which  would  fall  into  an 
ordinary  open  pail  are  kept  out  by  the  use  of  a  pail  of  the  type  shown  in 
No.  3,  figure  142.  The  following  figures  give  average  results  of  trials 
in  three  different  barns. 

BACTERIAL  COUNTS  OBTAINED  WITH  OPEN  AND  SMALL  TOP  PAILS 

Barn  Kind  of  pail  Average  bacterial  count 

f  Open  1,610 

\  Covered  280 

/    °PCn  6»000 

\  Covered  3,ooo 

/  Open  33,ooo 

'•  \  Covered  I>74° 

AVOID  DUST  IN  THE  ATMOSPHERE. — Many  of  the  necessary 
operations  of  the  cow  stable  stir  up  large  quantities  of  dust  and  fill  the 
air  with  microorganisms.  It  is  astonishing  to  see  how  many  bacteria 
can  adhere  to  a  small  piece  of  hay  or  may  be  found  in  a  gram  of  some 
of  our  common  dairy  feeds.  When  these  materials  are  fed  dry  just 
previous  to  milking  time,  the  atmosphere  of  the  stable  will  be  filled 
with  organisms  some  of  which  may  settle  into  the  milk  while  it  is  ex^ 
posed  during  the  process  of  milking.  The  effect  of  this  source  of  con- 
tamination in  one  stable  may  be  seen  by  the  following  experiments: 

BACTERIAL  CONTENT  OF  MILK  AS  AFFECTED  BY  FEEDING  DRY  HAY  AND  GRAIN 

Experiment  Date  Nature  of  sample     Number  bacteria  per 


No.  i 

i, 

Mav    4 

i  Before  feeding 

350 

No.  2      

May  17 

After   feeding 
Before  feeding 

2,900 

No.  3.. 

• 
May  18 

!  After    feeding 
Before  feeding 

4,4oo 
4,100 

•  After    feeding 

7,200 

In  another  stable*  where  the  sanitary  conditions  were  above  the  aver- 
age and  where  all  the  conditions  were  carefully  controlled,  the  atmos- 
phere added  from  7  to  937  germs  to  each  c.c.  of  the  milk,  the  number 
varying  with  the  amount  of  dust  in  the  air. 

*  X.  Y.  (Geneva)  Agr.  Exp.[Sta.'^Bull/40O. 


444  MICROBIOLOGY   OF   MILK   AND   MILK   PRODUCTS 

DAIRY  UTENSILS. — All  utensils  which  are  to  be  used  in  connection 
with  milk  should  be  so  constructed  that  there  are  no  cracks  or  crevices 
in  which  the  milk  can  accumulate  and  from  which  it  is  not  easily 
washed.  A  milk  pail  with  an  open  seam  may  be  the  cause  of  serious 
trouble  in  the  dairy.  The  dairy  utensils  should  be  simple  in  construc- 
tion, and  so  made  that  they  can  be  thoroughly  cleansed  with  ease 
and  made  of  such  material  that  they  can  be  thoroughly  sterilized 
either  with  water  which  is  actually  boiling  or  in  steam.  They  should 
then  be  thoroughly  dried  and  kept  so  till  again  needed  for  use.  When 
moisture  is  left  in  cans  and  other  utensils  bacteria  can  grow  rapidly 
and  be  the  means  of  serious  contamination  when  fresh  milk  is  poured 
into  them. 

THE  MILKER. — No  food  material  requires  greater  care  and  cleanli- 
ness on  the  part  of  those  handling  it  than  does  milk.  All  persons  having 
to  do  with  the  handling  of  this  delicate  food  product  should  constantly 
keep  in  mind  that  clean  hands  and  clothing  and  extreme  cleanliness  in 
every  operation  is  very  necessary  if  milk  of  good  quality  is  to  be  ob- 
tained. 

GROUPS  OR  TYPES  or  MICROORGANISMS  FOUND  IN  MILK  AND  THEIR 

SOURCES 

In  studying  the  types  of  bacteria  found  in  milk,  it  is  convenient 
to  arrange  them  in  groups  based  upon  their  action  on  the  milk  and 
their  effect  upon  persons  consuming  it.  There  are  certain  types  of 
organisms  which  are  very  troublesome  to  the  milk  dealer  but  which  are 
not  injurious  to  the  consumer.  Other  species  which  may  be  of  little  or 
no  significance  from  their  action  on  the  milk  are  of  greatest  significance 
from  the  standpoint  of  the  consumer  since  most  of  the  disease  organisms 
which  may  be  carried  by  milk  have  no  appreciable  action  upon  it.  Still 
other  forms  are  of  but  little  importance  to  either  the  dealer  or  the  con- 
sumer and  others  are  troublesome  to  both. 

GENERAL  SIGNIFICANCE  OF  ACID-FORMING  BACTERIA. — Of  all  the 
bacteria  that  find  their  way  into  milk,  those  that  are  able  to  ferment  the 
milk  sugar,  producing  from  it  different  kinds  and  amounts  of  acids,  find 
more  favorable  conditions  for  growth  at  ordinary  temperatures,  15°  to 
45°,  than  do  those  belonging  to  other  groups.  Because  of  their  greater 
rapidity  of  growth  and  because  of  the  inhibiting  effect  of  their  by-prod- 


THE   RELATION   OF   MICROORGANISMS   TO   MILK  445 

ucts  upon  the  other  groups  of  bacteria,  the  acid-forming  types  tend  to 
predominate  in  milk  and  the  specific  change  which  they  produce,  the 
souring,  is  of  such  common  occurrence  that  it  is  often  looked  upon  as 
something  inherent  in  milk. 

GROUPS  or  ACID-FORMING  BACTERIA.* — The  acid-forming  bacteria 
that  are  constantly  present  in  milk  represent  many  kinds  which  differ  in 
morphology,  in  cultural  characteristics,  and  in  their  products  of  fermen- 
tation. They  may  be  divided  into  four  groups  that  vary  greatly  as  far 
as  their  importance  in  the  handling  of  milk  is  concerned.  If  milk  is  pro- 
duced under  clean  conditions  and  is  kept  at  temperatures  ranging  from 
15°  to  35°,  the  acid  fermentation  will  be  almost  wholly  due  to  a  group  of 
bacteria  closely  allied  to  one  of  the  pathogenic  forms,  Strept.  pyogenes 
(Rosenbach).  To  representatives  of  this  group,  which  is  of  the  great- 
est importance  in  all  phases  of  dairying,  have  been  given  various  names 
by  different  investigator?.  The  most  important  organism  of  this  group 
is  one  to  which  the  name  Bact.  lactis  acidi  is  applied.  The  group  undoubt- 
edly includes  a  large  number  of  organisms,  all  of  which  produce,  how- 
ever, a  similar  change  in  milk. 

Second  in  importance  is  a  group  of  organisms,  of  which  the  best 
known  representatives  are  B.  coli  communis  and  Bact.  lactis  aerogenes. 
A  large  number  of  organisms  of  this  group  have  been  described  and 
named.  The  most  important  characteristics  of  the  representatives 
mentioned  will,  however,  suffice  to  characterize  the  group.  A  third 
group  is  represented  by  Bact.  bulgaricum  and  the  rod-shaped  organisms 
that  were  first  studied  in  detail  by  de  Freudenreich.  A  fourth  group 
includes  many  acid-forming  cocci,  some  of  which  exhibit  proteolytic 
properties  while  others  do  not.  Organisms  of  the  third  and  fourth 
groups  exert  little  or  no  effect  in  the  normal  acid  fermentation  of  milk, 
although  they  are  constantly  present  in  varying  numbers,  as  can  be 
demonstrated  by  appropriate  means,  and  are  of  importance  in  certain 
phases  of  dairy  manufacturing. 

In  any  sample  of  milk  the  relative  number  of  bacteria  belonging  to 
each  of  the  first  two  groups  is  dependent  upon  the  conditions  surround- 
ing production,  especially  with  reference  to  cleanliness.  The  bacteria 
belonging  to  the  first  group  come  largely  from  the  milk  utensils  and  are 
also  found  in  the  dust  of  the  barn  and  on  the  coat  of  the  animal.  The 
source  of  the  second  group  is  largely  the  fecal  matter  that  gains  entrance 
to  the  milk,  although  they  are  also  found  in  the  upper  layers  of  the  soil 

*  Prepared  by  E.  G.  Hastings. 


446  MICROBIOLOGY   OF   MILK   AND   MILK  PRODUCTS 

and  on  grain.  They  are  introduced  into  the  milk  with  the  dirt.  The 
cleaner  the  conditions  of  production,  the  smaller  will  be  the  number  of 
these  two  groups  of  organisms  found  in  fresh  milk. 

The  manufacture  of  the  leading  type  of  butter  and  of  all  kinds  of 
cheese  is  dependent  on  the  action  of  microorganisms,  hence  dairy  manu- 
facturing should  be  classed  as  a  true  fermentation  industry.  In  all 
such  industries  one  of  the  factors  determining  the  quality  of  the  product 
is  the  type  of  microorganism  employed  to  produce  the  desired  fermen- 
tation, and  the  importance  of  insuring  the  presence  of  desirable  organ- 
isms, and  the  exclusion  of  harmful  kinds  is  well  recognized. 

The  most  important  properties  of  organisms  employed  in  the  fermen- 
tation industries  are  the  physiological  rather  than  the  cultural  or  mor- 
phological, since  the  quality  of  the  product  is  dependent  on  the  by- 
products of  the  fermentation.  Hence  in  characterizing  the  groups  of 
acid-forming  bacteria,  the  biochemistry  of  each  group  will  be  empha- 
sized rather  than  the  cultural  and  morphological  characteristics  of  the 
members  of  the  group. 

Characteristics  of  the  Bact.  Lactis  Acidi  Group  * — The  organisms  of 
this  group  are  widely  distributed  in  nature,  as  is  shown  by  the  constancy 
with  which  milk  undergoes  the  characteristic  fermentation  produced  by 
the  members  of  the  group. 

The  cells  are  oval  in  form,  about  0.6 p,  to  i/z  in  length,  and  G.$IJI.  in 
diameter.  The  shorter  cells  appear  nearly  spherical,  which,  together 
with  the  fact  that  chains  of  cells  often  occur,  has  led  some  to  classify 
them  among  the  cocci  and  Kruse  has  applied  the  name  Strept.  lacticus  to 
a  member  of  the  group.  In  milk  the  cells  are  usually  in  twos,  the  outer 
ends  of  the  two  cells  being  pointed.  None  of  the  group  is  motile;  spores 
are  not  formed  and  capsules  are  often  noted.  The  members  of  the 
group  are  Gram-positive. 

The  optimum  temperature  for  growth  lies  between  30°  and  35°,  the 
minimum  growth  temperature  ranging  from  10°  to  12°,  while  the  maxi- 
mum is  42°.  They  are  to  be  classed  as  facultative  aerobes.  The  growth 
on  all  culture  media  is  marked  by  its  meagerness;  in  the  absence  of  a  fer- 
mentable carbohydrate,  no  growth  usually  occurs;  peptone  favors  the 
growth  even  in  milk.  In  the  case  of  freshly  isolated  cultures,  the 
growth  is  almost  invisible,  on  slopes  of  sugar  agar  appearing  as  small 
discrete  colonies.  On  sugar  agar  plates  the  colonies  are  small,  often 

*  Prepared  by  E.  G.  Hastings. 


THE  RELATION  OF  MICROORGANISMS  TO  MILK       447 

surrounded  by  a  hazy  zone,  and  always  occur  below  the  surface  of  the 
medium.  In  lactose-agar  stab  cultures  growth  occurs  along  the  entire 
line  of  inoculation,  but  there  is  no  surface  growth.  No  liquefaction  of 
gelatin  occurs.  In  bouillon  the  medium  is  uniformly  turbid  or  it  re- 
mains clear  with  a  slight  sediment.  On  potato,  growth  is  slight  or  is 
absent.  Milk  is  usually  curdled  within  twenty-four  hours  at  the  opti- 
mum temperature  by  members  of  the  group,  although  some  fail  to  cur- 
dle the  milk,  since  the  maximum  amount  of  acid  produced  is  not  suffi- 
cient to  cause  this  phenomenon.  Still  others  cause  curdling  in  the  pres- 
ence of  small  amounts  of  acids,  in  which  case  a  rennet-like  enzyme  may 
be  present.  No  gas  is  produced  in  the  fermentation  of  lactose,  hence 
the  curd  formed  in  milk  is  perfectly  homogeneous;  it  shows  but  little 
tendency  to  shrink  and  to  express  whey.  In  litmus  milk  the  color  is 
discharged  from  the  entire  mass  of  medium  before  curdling  occurs,  due 
to  the  reduction  of  the  litmus  to  the  colorless  leuco-compound.  Through 
the  action  of  the  oxygen  of  the  air  the  litmus  is  slowly  reoxidfzed  and 
the  pink  layer,  which  immediately  after  curdling  is  but  a  few  millimeters 
in  depth,  is  slowly  extended  until  the  entire  mass  of  curd  has  a  uniform 
pink  color.  Saccharose,  dextrose,  maltose,  and  mannit  are  fermented. 
The  maximum  amount  of  acid  produced  by  organisms  that  are  most 
typical  of  the  group  is  determined  by  the  composition  of  the  medium. 
It  is  often  said  that  the  organisms  causing  the  normal  souring  of  milk 
represent  a  group  that  can  grow  in  a  strongly  acid  medium.  This  is 
true  as  far  as  acid  salts  are  concerned,  but  free  acid  totally  inhibits 
growth.  In  a  culture  medium,  which  contains  no  substance  that  can 
combine  with  the  acid  formed  and  thus  remove  it  from  the  sphere  of 
action,  no  growth,  or  but  very  slight  growth  occurs.  In  sugar  bouillon 
and  in  milk,  the  amount  of  acid  formed  is  determined  by  the  amount  of 
substances  in  these  liquids  that  can  combine  with  the  acid.  In  milk 
such  compounds  are  the  casein  and  some  of  the  ash  constituents, 
especially  the  phosphates.  In  normal  milk,  the  maximum  acidity 
attained  ranges  from  0.9  to  1.25  per  cent  calculated  as  lactic  acid.  If 
the  content  of  neutralizing  compounds  per  unit  volume  is  varied  by 
concentration,  dilution,  or  by  the  addition  of  such  substances  as  cal- 
cium phosphate,  the  maximum  amount  of  acid  produced  by  typical 
cultures  will  be  changed.  In  sugar  bouillon  the  maximum  acidity 
produced  rarely  exceeds  0.25  per  cent. 


448 


MICROBIOLOGY   OF   MILK  AND   MILK   PRODUCTS 


The  fermentation  of  lactose  is  usually  expressed  as  follows: 
Ci2H22On  +  H2O  =  4C3H6O3. 

Thus  342  parts  of  lactose  should  yield  360  parts  of  lactic  acid.  The 
theoretical  yield  of  lactic  acid  is  never  obtained,  for  the  action  of  the 
organism  on  the  carbohydrate  is  much  more  complex  than  is  represented 
by  the  equation  given.  In  the  following  table  are  given  data  obtained 
by  a  number  of  investigators. 

These  data  signify  that  other  compounds  than  lactic  acid  are 
formed  in  the  fermentation  of  lactose  by  these  acid-forming  bacteria. 
Acetic  acid  (CH3.COOH);  formic  acid  (H.COOH);  propionic  acid 


[Sugar  content  of 
milk, 
per  cent. 

Sugar  fermented, 
per  cent. 

Lactic  acid  calcu- 
lated, 
per  cent. 

Lactic  acid  found, 
per  cent,  of  theo- 
retical 

4-54 
4.96 

4-94 

O.6o 
0.56 
0.65 

0.632 
0.590 
o  684 

8Q.56 
98.13 
97.89 

(C2H5.COOH);  traces  of  alcohols,  aldehydes  and  esters  have  been 
found.  The  lactic  acid  formed  is  the  dextro  modification.  It  is  be- 
lieved that  the  fermentation  is  due  to  an  enzyme,  lactacidase,  one  of  the 
intracellular  enzymes  that  can  be  demonstrated  only  with  difficulty. 

Milk  fermented  by  members  of  this  group  has  a  mild  acid  taste,  an 
agreeable  odor,  and  the  curd  can  be  so  finely  divided  by  agitation  as  to 
produce  almost  as  perfect  an  emulsion  as  in  raw  milk.  The  organisms 
are  to  be  classed  as  desirable  from  the  standpoint  of  the  dairy  manu- 
facturer, and  the  fermentation  produced  by  them  may  be  called  a  true 
lactic  fermentation. 

Characteristics  of  the  B.  Coli-aero genes  Group.* — This  group  includes 
a  considerable  variety  of  organisms,  which  differ  in  morphology,  in  cul- 
tural characteristics  and  undoubtedly  in  the  character  and  amounts  of 
their  by-products.  They  are  more  distinctly  bacilli  than  the  members 
of  the  preceding  group;  are  motile  or  non-motile;  none  produces  spores 
and  they  are  usually  negative  to  Gram's  stain.  The  optimum  growth 
temperature,  35°  to  40°,  is  somewhat  higher  than  for  the  preceding 

*  Prepared  by  E.  G.  Hastings. 


THE    RELATION    OF   MICROORGANISMS    TO   MILK  449 

group,  the  vegetation  range  being  15°  to  45°.  They  are  to  be  classed  as 
facultative  anaerobes. 

The  conditions  for  development  are  less  narrow  than  for  the  Bact. 
lactis  acidi  group,  growth  occurring  on  all  the  ordinary  culture  media 
and  in  the  absence  of  carbohydrates.  -  Indol  and  hydrogen  sulphide 
are  often  formed  and  nitrates  are  reduced.  The  growth  is  usually  pro- 
fuse, the  colonies  large  and  surface  growth  occurring  in  stab  cultures. 
Gelatin  is  not  usually  liquefied. 

Lactose,  dextrose  and  saccharose  are  fermented,  with  the  production 
of  varying  amounts  of  gas  in  which  have  been  found  carbon  dioxide, 
hydrogen  and  methane.  The  maximum  amount  of  acid  produced  in 
any  culture  medium  is  quite  similar  to  that  formed  by  the  members  of 
the  previous  group.  The  relative  proportions  between  the  non- volatile 
and  volatile  acids  are  far  different,  lactic  acid  comprising  less  than  30 
per  cent  of  the  total  acid  formed  while  volatile  acids,  such  as  acetic 
and  formic,  make  up  the  remainder.  Traces  of  succinic  acid 
(C2H4(COOH)2)  and  alcohol  have  also  been  found.  The  lactic  acid  is 
of  the  laevo-form. 

Milk  is  usually  curdled,  although  some  members  of  the  group  do  not 
produce  enough  acid  to  cause  curdling.  The  amount  of  gas  produced 
varies  widely.  In  the  case  of  those  forms  that  cause  curdling,  the 
presence  of  gas  is  made  evident  by  rents  in  the  curd.  If  consider  ble 
gas  is  produced,  the  curd  will  be  very  spongy.  When  the  acid  formed  is 
not  sufficient  to  curdle  the  milk,  the  gas  produced  is  likely  to  pass  off 
unnoticed.  The  curd  shrinks  to  a  greater  or  less  extent  and  thus 
becomes  so  firm  that  it  is  impossible  to  emulsify  it  again.  The  odor  of 
the  fermented  milk  is  often  offensive  and  the  taste  disagreeable  and 
sharp.  The  organisms  of  this  group  are  to  be  classed  as  undesirable 
and  the  fermentation  produced  by  them  cannot  correctly  be  called 
a  lactic  fermentation. 

Representatives  of  these  two  great  groups  of  acid-forming 
bacteria  are  to  be  found  in  every  sample  of  market  milk  in  varying 
proportions.  Both  find  in  milk  favorable  conditions  for  growth,  and 
the  normal  souring  is  produced  conjointly  by  them,  each  producing 
its  own  specific  products,  the  relative  amounts  of  which  are  largely 
dependent  on  the  number  of  each  group  that  is  originally  introduced 
into  the  milk  and  on  the  temperature  at  which  it  is  kept.  The  higher 
temperatures  tend  to  favor  the  growth  of  members  of  the  B.  coli- 
29 


450  MICROBIOLOGY    OF   MILK   AND    MILK   PRODUCTS 

aerogenes  group  over  that  of  the  Bact.  lactis  acidi  group.  The  value  of 
milk  for  butter  and  cheese  is  determined  by  the  relative  amounts  of 
the  products  of  the  desirable  and  the  undesirable  acid-forming  bacteria. 

The  difference  in  taste  and  odor  between  milk  fermented  by  pure 
cultures  of  Bact.  lactis  acidi,  and  that  which  has  soured  spontaneously, 
emphasizes  the  difference  in  the  products  of  the  fermentations  produced 
by  the  two  groups  of  acid-forming  bacteria. 

Characteristics  of  the  Bact.  Bulgaricum  Group* — The  organisms  of 
this  group  are  to  be  classed  as  true  lactic  bacteria,  since  they  produce 
almost  exclusively  lactic  acid  from  the  sugar  fermented  and  only  small 
quantities  of  other  acids  as  formic,  acetic,  and  propionic.  They  vary 
widely  in  form  and  size;  but  are  usually  large  rods,  2^1  to  $fj.  long  and 
0.5/1  to  in  wide.  There  is  a  tendency  to  form  long  threads.  They 
are  Gram-positive  and  when  stained  with  methylene  blue  often  show 
distinct  granules  in  the  cells;  with  Neisser's  stain  the  appearance  of 
some  cultures  is  similar  to  that  of  the  diphtheria  bacterium.  They 
are  non-motile  and  do  not  form  spores;  capsules  are  seldom  noted.  The 
optimum  growth  temperature  is  from  40°  to  50°  and  the  minimum  is 
asserted  to  be  25°,  although  for  many  members  of  the  group  it  must  be 
much  lower. 

The  growth  on  all  ordinary  culture  media  is  meager  or  is  absent; 
the  colonies  are  often  microscopic  in  size  and  show  radiating  threads. 
Free  acids  do  not  inhibit  development  and  the  term  acidophilous  has 
been  applied  to  the  group.  They  grow  slowly  in  milk,  even  at  the 
optimum  temperature,  and  curdling  may  not  occur  for  several  days; 
the  curd  is  homogeneous  and  in  litmus  milk  reduction  occurs.  The 
maximum  amount  of  acid  varies  from  1.25  to  4.0  per  cent.  Some 
members  of  the  group  produce  dextro-,  others  laevo-acid,  and  racemic 
acid  is  formed  in  some  cases.  The  curd  may  be  easily  broken  by  agita- 
tion, and  through  the  solvent  action  of  the  acid  is  partially  dissolved. 
The  organisms  do  not  liquefy  gelatin,  but  the  casein  of  milk  is  partially 
changed  into  soluble  decomposition  products,  as  was  first  shown  by  de 
Freudenreich,  and  later  confirmed  by  Hastings. 

It  has  been  supposed  by  many  that  this  group  was  confined  to 
and  characteristic  of  certain  of  the  fermented  milks,  especially  those 
of  eastern  Europe  and  western  Asia,  such  as  Yogurt  and  Matzoon. 
Recent  work  has  shown  that  this  group  is  widely  distributed  in  nature. 

*  Prepared  by  E.  G.  Hastings. 


THE   RELATION   OF   MICROORGANISMS    TO   MILK  45! 

Representatives  of  this  group  are  found  constantly  in  milk  and  other 
dairy  products.  Their  presence  in  milk  can  be  demonstrated  by 
placing  a  sample  of  milk  in  a  corked  bottle,  and  incubating  at  37°.  The 
acidity  of  the  milk  increases  rapidly  at  first,  due  to  the  growth  of  the 
members  of  the  two  previous  groups.  These  ordinary  acid-forming 
organisms  are  soon  inhibited  by  the  appearance  of  free  acid,  but  the 
acidity  of  the  milk  nevertheless  continues  to  increase  slowly,  and 
with  this  continued  increase  a  change  in  flora  is  noted,  the  short, 
plump  bacilli  ceasing  to  predominate  and  long  slender  rods  constantly 
increasing  in  numbers.  The  source  of  this  group  is  undoubtedly 
the  alimentary  tract  of  the  animal. 

Characteristics  of  the  Coccus  Group* — This  group  is  well  represented 
by  the  bacteria  which  form  the  characteristic  flora  of  the  udder.  They 
vary  greatly  in  size  and  in  other  properties.  They  retain  Gram's 
stain;  many  are  chromogenic,  the  color  ranging  from  a  white  to  a 
deep  orange.  They  grow  slowly  on  all  ordinary  culture  media,  but 
the  growth  is  not  necessarily  meager.  Generally  they  are  aerobic, 
although  many  grow  under  anaerobic  conditions.  Gelatin  may  be 
liquefied  or  not.  Milk  may  or  may  not  be  curdled,  the  curd  often 
resembling  that  formed  by  rennet-like  enzymes.  They  produce  no 
lactic  acid,  but  only  acetic,  propionic,  butyric  and  caproic  acids, 
and  hence  cannot  be  classed  as  lactic  bacteria. 

BACTERIA  HAVING  No  APPRECIABLE  EFFECT  ON  MILK. — This 
group  is  made  up  of  many  different  forms.  They  produce  no  changes, 
during  the  normal  life  of  market  milk,  which  can  be  detected  either 
by  the  eye  or  the  taste.  They  do  not  develop  very  rapidly  in  milk, 
and  some  species  gradually  disappear  while  others  increase  in  numbers. 
Many  of  the  organisms  in  this  group  are  chromogenic,  orange  and 
lemon  yellows  being  among  the  more  common  forms.  They  are 
mostly  cocci  and  do  not  liquefy  gelatin.  From  the  standpoint  of  the 
commercial  milkman  these  organisms  are  of  little  significance  and  this 
is  probably  also  true  from  the  standpoint  of  the  consumer. 

THE  CASEIN-DIGESTING  OR  PEPTONIZING  BACTERIA. — These  organ- 
isms digest  the  casein  either  with  or  without  coagulation.  Many  of 
them  cause  the  milk  to  curdle.  The  reaction  is  alkaline.  The  curdling 
agent  is  a  rennet-like  enzyme.  They  liquefy  gelatin.  Most  of  the 
organisms  of  this  group  are  rods  of  various  shapes  and  sizes,  some 

*  Prepared  by  E.  G.  Hastings 


452  MICROBIOLOGY   OF    MILK   AND    MILK   PRODUCTS 

of  them  being  the  largest  rods  found  in  milk.  Some  are  motile  and 
some  non-motile.  Some  representatives  of  this  group  produce  little 
or  no  odor,  but  many  of  the  species  develop  very  strong  putrefactive 
odors.  Barny  or  cowy  odors  or  other  off-flavors  sometimes  found  in 
milk  and  dairy  products  may  be  caused  by  the  action  of  this  type 
of  bacteria.  They  are  associated  with  filth  and  their  presence  in 
milk  indicates  insanitary  conditions  of  production  or  handling. 

PATHOGENIC  ORGANISMS. — This  group  includes  all  those  species 
which  may  gain  access  to  milk,  which  are  capable  of  causing  specific 
diseases  in  human  beings.  They  are  of  the  greatest  importance  to  the 
consumer.  They  do  not  appreciably  affect  the  physical  or  chemical 
properties  of  the  milk,  or  produce  any  changes  in  its  appearance, 
flavor,  or  keeping  quality  which  would  indicate  their  presence. 
Some  of  them  do  not  even  develop  in  milk,  as  is  the  case  with  the  Bad. 
tuberculosis.  Others,  as  the  diphtheria  bacteria  and  typhoid  fever 
bacilli,  may  grow  in  milk  with  great  rapidity.  This  group  also  con- 
tains certain  species  which  produce  diarrhceal  disorders,  especially 
in  infants  and  young  children.  Some  of  them  are  probably  organisms 
which  are  also  included  in  the  peptonizing  group.  The  specific 
pathogenic  organisms,  possibly  with  the  exception  of  Bad.  tubercu- 
losis, get  into  milk,  either  directly  or  indirectly,  from  human  patients 
suffering  with  the  particular  disease. 

FACTORS  INFLUENCING  THE  DEVELOPMENT  OF  MICROORGANISMS  IN 

MILK 

The  number  of  microorganisms  found  in  fresh  milk  shows  its  bac- 
terial condition  at  that  time,  but  it  gives  little  idea  of  the  organisms 
which  may  be  found  in  the  same  milk  at  later  periods.  There  are 
many  factors  to  be  considered  if  we  wish  to  study  the  development 
of  the  various  types  which  get  into  ordinary  milk.  These  factors 
may  be  considered  briefly  under  the  following  heads: 

INITIAL  CONTAMINATION. — Fresh  milk  varies  widely  in  the  number 
of  organisms  which  it  contains  as  a  result  of  the  conditions  under 
which  it  has  been  produced.  There  are  differences  not  only  in  the 
numbers  of  organisms  but  also  in  the  species  which  may  be  found  in 
different  samples  of  fresh  milk.  Both  of  these  factors  are  important 
in  the  later  changes  which  may  take  place.  The  effect  of  numer- 
ical initial  contamination  may  be  seen  in  the  following  tables  where 


THE   RELATION   OF   MICROORGANISMS   TO   MILK 


453 


EFFECT  OF  INITIAL  CONTAMINATION  ON  DEVELOPMENT  OF  BACTERIA  AND  KEEPING 

QUALITY  OF  MILK 

Milk  Having  Moderately  High  Initial  Contamination 


Bacteria  per  c.c.  in  fresh 
milk 

Bacteria  12  hours 

Bacteria  36  hours 

Hours  to  curdling 

187,000 

432,000 

633,500,000 

45 

Milk  Having  Moderate  Initial  Contamination 


Bacteria  per  c.c.  in  fresh 
milk 

Bacteria  12  hours 

Bacteria  36  hours 

Hours  to  curdling 

3,000 

14,000 

149,650,000 

99 

Milk  Having  Small  Initial  Contamination 


Bacteria  per  c.c.  in  fresh 
milk 

Bacteria  12  hours 

Bacteria  36  hours 

Hours  to  curdling 

325 

1,712 

10,125,000 

121 

milk  starting  out  with  different  numbers  of  organisms  was  kept  under 
similar  conditions  until  coagulation.  Plate  cultures  made  from  these 
three  samples  show  the  relative  development  of  the  number  of 
organisms. 

These  samples  were  all  kept  at  a  constant  temperature  of  21°  and 
the  difference  in  the  numbers  of  bacteria  and  the  curdling  time  can 
therefore  be  fairly  attributed  to  the  difference  in  the  initial  contamina- 
tion of  the  three  samples.  All  three  of  the  samples  showed  a  normal 
development  of  the  lactic  organisms,  which  constituted  over  99  per 
cent  of  the  total  organisms  present  at  the  time  of  curdling.  While 
this  may  be  considered  as  showing  the  normal  effect  of  the  original 
contamination  upon  the  milk,  it  is  well  to  bear  in  mind  the  fact  that 
there  are  many  apparent  exceptions  due  to  some  particular  type  of 
organism  predominating  and  interfering  with  the  normal  development 
of  the  lactic  types. 

STRAINING. — The  straining  of  milk  is  one  of  the  most  common 
operations  in  connection  with  its  handling  and  is  considered  by  most 
dairymen  as  one  of  the  most  essential  from  the  standpoint  of  the  qual- 


454 


MICROBIOLOGY  OP   MILK  AND   MILK  PRODUCTS 


ity  of  the  milk.  If  milk  is  strained  through  cheese  cloth  or  wire 
gauze  much  of  the  insoluble  dirt  can  be  removed.  This  has  led  to  the 
general  belief  that  straining  improves  the  sanitary  and  keeping  quali- 
ties of  the  milk. 

The  effect  of  straining  on  removal  of  insoluble  dirt  is  shown  by 
the  following  results  of  tests: 

DIRT  REMOVED  BY  PASSING  MILK  THROUGH  Two  THICKNESSES  OF  FINE  CLOTH 
(Weight  of  insoluble  dirt  given  in  milligrams  per  liter  of  milk) 


Experiment 

Before  straining 

After  straining 

Per  cent  removed 

No   i 

8   OS 

4    7O 

4.7    S 

No.  2  

95 
$    SS 

4.70 

4..QC 

10.8 

No  * 

C      1C 

2    OS 

4.2    7 

No.  4 

2    4C 

O.  2O 

01.8 

No   q 

r    ne 

3IO 

38  6 

It  may  be  noticed  that  even  after  straining  the  milk  contained 
appreciable  quantities  of  insoluble  dirt  which  had  passed  through 
the  strainer  cloth.  The  difference  in  per  cent  of  dirt  removed  in 
different  samples  is  due  to  the  nature  of  the  dirt  itself.  The  coarser 
the  dirt  the  greater  the  proportion  that  will  be  removed  by  straining. 

It  is  not  true,  however,  that  the  keeping  quality  is  necessarily 
improved  by  the  simple  process  of  straining.  It  depends  largely  upon 
the  condition  of  the  miJk  and  the  nature  of  the  strainer.  Not  infre- 
quently passing  milk  through  a  strainer  not  only  fails  to  improve  its 
keeping  quality  but  actually  injures  it.  This  has  been  shown  by  a 
number  of  investigators.  The  effect  of  straining  upon  the  germ  con- 
tent may  be  seen  in  the  following  figures  where  the  milk  was  passed 
through  a  strainer  composed  of  three  thicknesses  of  fine  cheese  cloth 
supported  by  wire  gauze. 

EFFECT  OF  STRAINING  UPON  BACTERIAL  CONTENT  OF  MILK 


Experiment 

Before  straining, 
bacteria  per  c.c. 

After  straining, 
bacteria  per  c.c. 

No 

I  

3,6oo 

3,600 

No 

2..                                                         . 

7,4OO 

6,900 

No 

12  800 

IO,SOO 

No 

8,800 

II.-Z7C 

No. 

4.  .  . 

S.  . 

2,800 

2,700 

THE    RELATION    OF   MICROORGANISMS    TO   MILK 


455 


The  effect  of  straining  upon  the  keeping  quality  is  shown  in  the 
following  experiments  where  the  milk  was  strained  through  the  same 
form  of  strainer  mentioned  above  and  the  samples  kept  at  constant 
temperature  of  21°  until  coagulation. 

EFFECT  OF  STRAINING  UPON  KEEPING  QUALITY  OF  MILK 


Not  strained, 
hours  to  coagulation 

Strained, 
hours  to  coagulation 

Experiment  No.  i  

42 

4.2 

Experiment  No.  2  

c;7 

cc 

Experiment  No.  3  

•7tr 

•2C 

Experiment  No  4 

80 

CA 

Experiment  No.  5  

tro 

"\O 

It  will  be  seen  that  in  no  case  was  the  keeping  quality  of  these 
samples  increased  by  the  straining  process  while  in  some  cases  it 
was  materially  injured. 

Cotton  filters  are  more  efficient  than  cheese  cloth  and  in  some 
cases  the  keeping  quality  of  the  milk  may  be  improved  by  this  process. 

AERATION. — This  is  the  process  of  exposing  the  milk  to  the  atmos- 
phere by  allowing  it  to  run  over  the  surface  of  the  aerator  in  a  very 
thin  film  If  milk  has  been  produced  under  such  conditions  that  it 
has  absorbed  foreign  odors,  this  process  may  be  of  value  in  getting 
rid  of  the  absorbed  odors,  but  from  the  bacterial  standpoint  the  process 
of  aerating  is  not  desirable,  since  it  gives  one  more  opportunity  for 
the  milk  to  become  contaminated  with  organisms  from  the  atmos- 
phere and  from  the  aerator  itself.  It  is  possible  to  aerate  milk  under 
such  conditions  that  the  germ  content  will  not  be  increased,  but  if 
aeration  takes  place  in  the  cow  stable  or  other  place  where  the  atmos- 
phere contains  dust  the  number  of  organisms  will  be  greater  after 
aeration  than  before,  the  amount  of  increase  being  proportional 
to  the  sanitary  conditions  under  which  the  aeration  is  done.  It  is 
even  possible  that  the  milk  may  absorb  foreign  odors  during  the  proc- 
ess of  aeration  and  be  of  poorer  quality  than  it  was  before.  It  is  thought 
by  many  that  the  process  of  aeration  is  necessary  in  order  to  get  rid 
of  the  so-called  animal  odors  commonly  found  in  milk.  These  odors 
are,  however,  not  normal  to  the  milk  but  are  absorbed  from  the  foul 
air  in  the  stables  or  other  sources.  This  is  shown  by  the  fact  that  some 
of  the  very  finest  quality  of  certified  milk  is  bottled  while  still  con- 


456 


MICROBIOLOGY    OF   MILK   AND    MILK   PRODUCTS 


taining  the  animal  heat  with  the  least  possible  exposure  to  the  air, 
tightly  sealed  at  once  and  plunged  into  ice  water.  Such  milk  contains 
no  suggestion  of  animal  odor.  Aeration  may  be  of  value  in  removing 
undesirable  odors  from  milk  which  is  not  produced  under  good 
sanitary  conditions,  if  done  in  an  atmosphere  free  from  all  dust  and 
odors,  but  it  is  not  necessary  for  milk  of  good  quality.  The  common 
belief  that  aeration  is  valuable  is  probably  due  to  the  fact  that  most 
aerators  are  coolers  as  well,  and  the  beneficial  results  are  due  to  the 
cooling  and  not  the  aeration. 

CENTRIFUGAL  SEPARATION. — It  is  a  common  practice  in  some  milk 
plants  to  pass  the  milk  through  a  centrifugal  separator  or  clarifier  to 
remove  any  dirt  which  it  may  contain.  This  operation  is  effective 
for  the  removal  of  much  of  the  insoluble  dirt  which  may  be  in  the  milk, 
but  it  is  of  doubtful  value  from  the  standpoint  of  the  bacterial  content 
and  the  keeping  quality  of  the  milk.  In  spite  of  the  fact  that  the 
separator  slime  is  very  rich  in  bacteria,  the  milk  and  cream  as  they 
come  from  the  machine  will  normally  show  larger  bacterial  counts  in 
agar  and  gelatin  plates  than  will  the  milk  before  treatment,  due  of 
course  to  the  breaking  up  of  the  bacterial  groups.  In  some  cases, 
however,  there  is  an  apparent  decrease.  The  usual  effect  upon  the 
germ  content  of  passing  milk  through  a  separator  or  clarifier  may  be 
seen  in  the  following  tables: 

INFLUENCE  OF  PASSING  MILK  THROUGH  A  CENTRIFUGAL  SEPARATOR  UPON  THE 
GERM  CONTENT  OF  THE  SKIM  MILK  AND  CREAM 


Plate  count  in 
whole  milk 

Plate  count  in 
skim  milk 

Plate  count  in  cream 

Sample  No  i 

•7Q  OOO 

69  ooo 

7<?  OOO 

Sample  No.  2  

44  OOO 

76,000 

70O,OOO 

Sample  No  3 

<\6  ooo 

7"?  OOO 

820  ooo 

Sample  No.  4  

200,000 

336.OOO 

330,000 

EFFECT   OF   A    CENTRIFUGAL    CLARIFIER   UPON   THE  GERM  CONTENT  OF  MILK 


Sample 
number 

Plate  count  before 
clarifying 

Plate  count  after 
clarifying 

Numerical 
increase 

Percentage 
increase 

I 

6,000 

9,000 

3,000 

50 

2 

15,000 

22,000 

7,000 

46 

3 

6o,OOO                    156,000 

96,000 

160 

4 

133,000                     197,000 

64,000 

48 

5 

370,000 

643,000                        273,000 

73 

THE   RELATION   OF   MICROORGANISMS   TO  MILK 


457 


Similar  results  have  been  reported  by  Bahlman,*  by  Mclnerney,f 
and  by  Sherman.  {  Some  investigators,  especially  Hammer ||  and 
Marshall  and  Hood,  §  have  reported  results  showing  that  in  some  lots 
of  milk  the  plate  count  from  the  clarified  milk  is  less  than  in  the  original 
milk.  This  is  shown  in  the  following  data  given  by  the  last  named 
authors. 

BACTERIA  IN  COMMERCIAL  MILK  BEFORE  AND  AFTER  CLARIFICATION 


Sample  No. 

Number  of  bacteria 
in  i  cubic  centi- 
meter of  un- 
clarified  milk 

Number  of  bacteria 
in  i  cubic  centi- 
meter of 
clarified  milk 

Per  cent  increase 

I 

250,000 

900,000 

260 

2 

100,000 

200,000 

100 

3                                   75,000 

65,000 

-13 

4                                     20,000 

50,000 

150 

5                                     5,000 

12,000 

14 

6                                125,000 

7O,OOO 

-44 

7                                130,000 
8                                  25,000 

400,000 
48,OOO 

207 
92 

9                                   20,000 

35,ooo 

75 

10                                                350,000 

250,000 

-28 

ii 

30,000 

40,000 

33 

12 

40,000 

50,000 

25 

13 

30,000 

20,000 

-33 

14 

10,000 

10,000 

15 

16,000 

33,000 

106 

In  the  case  of  the  increased  counts  they  do  not  mean  that  there  is  an 
actual  increase  in  individual  bacteria  in  these  samples  due  to  the  action 
of  the  separator  or  clarifier.  What  it  does  mean  is  that  the  small 
clusters  or  groups  of  organisms,  as  they  exist  in  the  whole  milk  are 
thrown  apart  by  the  centrifugal  force  and  therefore  develop  a  larger 
number  of  individual  colonies  in  the  plate  cultures  in  spite  of  the  fact 
that  large  numbers  of  organisms  are  thrown  out  in  the  slime. 

*  Bahlman,  Clarence.  Milk  Clarifiers,  Am.  Jour,  of  Public  Health,  1916,  Vol.  VI,  No.  8, 
1916. 

t  Mclnerney,  T.  J.     Clarification  of  Milk.     Cornell  Agr.  Exp.  Sta.  Bull.  389,  April,  1917. 

J  Sherman,  James  M.  Bacteriological  Tests  of  Milk  Clarifier.  Jour,  of  Diary  Science, 
1917,  Vol.  I,  No.  3,  p.  272. 

||  Hammer,  B.  W.  Studies  on  the  Clarification  of  Milk.  Iowa  Agr.  Exp.  Sta.  Bull.  28, 
1916. 

§  Marshall,  C.  E.  and  Hood,  E.  G.  Clarification  of  Milk.  Mass.  Agr.  Exp.  Sta.  Bull.  187, 
Nov.,  1918. 


458 


MICROBIOLOGY   OF  MILK  AND   MILK  PRODUCTS 


TEMPERATURE. — The  temperature 'at  which  milk  is  kept  is  one  of 
the  most  important  factors  determining  the  development  of  its  micro- 
bial  content.  Every  one  at  all  familiar  with  milk  knows  that  it  spoils 
very  quickly  if  allowed  to  stand  at  warm  temperatures.  If,  how- 
ever, the  milk  is  held  at  temperatures  of  10°  or  lower,  its  keeping 
quality  is  greatly  increased.  Most  of  the  ordinary  species  of  organisms 
which  gain  entrance  to  milk  do  not  grow  rapidly  at  temperatures 
of  10°  or  lower.  There  are,  however,  certain  species  which  will 
grow  with  considerable  rapidity  at  temperatures  below  10°,  especially 
some  of  the  spore-bearing  non-acid  forms.  If  the  temperature  of  the 
milk  is  allowed  to  rise  above  10°,  the  growth  of  the  common  species 
increases  rapidly.  The  influence  of  temperature  upon  the  develop- 
ment of  bacteria  may  be  seen  in  the  following  experiment  where 
a  given  lot  of  milk  was  thoroughly  mixed  and  divided  into  seven 
portions,  which  were  then  held  at  the  temperatures  indicated  for 
twelve  hours,  at  the  end  of  which  time  they  were  plated  for  the 
total  germ  content. 

EFFECT  OF  DIFFERENT  TEMPERATURES  UPON  THE  DEVELOPMENT  OF  BACTERIA 

IN  MILK 


Temperature 
for  12 

maintained 
hours 

Plate  count  per  c.c.  at  end 
of  12  hours 

Hours  to  curdling 
at  21° 

c. 

F. 

4.5° 

40° 

4,000 

75 

7° 

45° 

9,000 

75 

10° 

50° 

18,000 

72 

12.5° 

55° 

38,000 

49 

15-5° 

60° 

4S3,ooo 

43 

21° 

70° 

8,800,000 

32 

26.5° 

80° 

55,300,000 

28 

The  fresh  milk  showed  a  count  of  5,000  per  c.c.  and  curdled  in 
fifty- two  hours  at  a  temperature  of  21°.  The  curdling  time  of  these 
samples  was  determined  by  placing  them  at  a  constant  temperature 
of  21°  at  the  close  of  the  twelve-hour  period  and  holding  them  at  this 
temperature  until  coagulation  took  place.  The  difference  in  time  of 
curdling  therefore  is  due  to  the  maintenance  of  the  special  tempera- 
ture for  twelve  hours  only  and  not  for  the  entire  period  up  to  the  time 
of  curdling. 


THE   RELATION   OF  MICROORGANISMS   TO   MILK  459 

PASTEURIZATION. — The  term  pasteurization  is  used  to  designate 
the  process  of  heating  milk  to  a  temperature  sufficient  to  destroy 
a  portion  of  the  bacteria,  including  the  pathogens,  and  then  cooling  it 
to  a  temperature  which  will  prevent  the  rapid  development  of  the 
organisms  that  are  left.  The  temperatures  commonly  used  for  this 
purpose  vary  from  60°  to  85°.  The  length  of  time  the  milk  is  exposed 
to  the  high  temperature  may  also  vary  from  a  few  seconds  to  thirty 
minutes,  depending  upon  the  method  employed.  The  two  chief 
purposes  for  the  pasteurization  of  milk  are  to  destroy  any  pathogenic 
organisms  which  the  milk  may  contain  and  t6  increase  its  keeping 
quality.  The  purpose  for  which  the  pasteurization  is  done  will  deter- 
mine the  method  used.  In  commercial  pasteurization,  where  the  chief 
purpose  is  to  destroy  the  lactic  organisms  and  thus  improve  the  keeping 
quality  of  the  milk,  the  method  used  is  that  known  as  the  "flash"  or  in- 
stantaneous method,  where  the  milk  is  subjected  to  a  high  temperature 
for  a  few  seconds  only  and  then  cooled.  In  this  method  of  pasteuriza- 
tion varying  degrees  of  efficiency  are  obtained,  depending  upon  a 
number  of  factors,  chiefly  the  bacterial  condition  of  the  milk  to -be 
pasteurized,  the  degree  of  heat  and  the  length  of  the  exposure  and  the 
temperature  to  which  the  milk  is  cooled.  By  this  method,  it  is  possible 
to  destroy  a  large  percentage  of  the  organisms  in  the  raw  milk,  and 
materially  increase  its  keeping  quality,  but  the  temperature  and  time  to 
which  any  particle  of  milk  is  exposed  cannot  be  accurately  controlled, 
and  this  method  cannot  be  depended  upon  to  kill  all  of  the  disease-pro- 
ducing organisms  which  may  be  in  the  milk.  This  method  has  been 
largely  abandoned  for  the  pasteurization  of  market  milk. 

Under  present  conditions  of  the  market  milk  business  where  the 
chief  purpose  of  pasteurization  is  to  render  the  milk  free  from  disease- 
producing  organisms,  the  so-called  "holding"  method  is  employed. 
This  consists  in  raising  the  temperature  of  the  milk  to  about  60° 
to  63°  and  holding  it  at  this  temperature  for  a  period  of  twenty  to 
thirty  minutes.  If  this  method  is  properly  done,  most  of  the  organisms 
except  certain  spore  forms  should  be  killed  and  the  milk  at  the  end  of 
the  pasteurizing  process  contain  only  a  small  percentage  of  its  original 
germ  content. 

Formerly  it  was  believed  that  heating  milk  to  a  high  temperature 
killed  all  the  lactic  acid  organisms,  and  favored  the  subsequent  growth  of 
other  more  undesirable  species,  but  more  recent  studies  on  the  bacterial 


460  MICROBIOLOGY   OF   MILK   AND   MILK   PRODUCTS 

flora  of  milk,  pasteurized  by  the  " holding"  method,  have  shown  that 
some  strains  of  the  lactic  acid  bacteria  can  survive  the  relatively  lower 
temperatures  used  in  this  method,  and  that  the  later  development  of 
the  different  groups  of  bacteria  is  similar  to  that  in  raw  milk  of  equal 
bacterial  grade. 

Pasteurization  at  the  temperatures  used  in  the  holding  process  does 
not  seem  to  cause  any  injurious  chemical  changes  in  the  milk  constitu- 
ents, or  affect  its  digestibility. 

Proper  pasteurization  gives  a  valuable  means  of  rendering  the  milk 
supply  for  our  cities  reasonably  free  from  pathogenic  microorganisms, 
but,  in  order  to  insure  this  safety,  the  work  must  be  carefully  done, 
and  all  later  contamination  avoided.  Preferably,  the  work  should  be 
done  under  expert,  municipal  supervision.  Undoubtedly  the  ideal 
method  is  pasteurization  in  the  sealed  bottle  which  is  to  be  delivered  to 
the  consumer,  since  this  method  reduces  to  the  minimum  the  danger 
of  subsequent  contamination. 

Pasteurization  must  not  be  regarded  as  a  substitute  for  care  and 
cleanliness  or  a  means  of  renovating  old  or  dirty  milk  otherwise  unfit 
for  use,  but  rather  as  an  additional  means  of  protecting  the  consumer 
against  disease-producing  microorganisms  in  the  milk  supply. 

THE  USE  OF  CHEMICALS. — The  addition  of  certain  chemicals  to  milk 
will  retard  the  growth  of  bacteria.  The  chemicals  most  commonly  used 
for  this  purpose  are  calcium  hypochlorite,  borax  and  formalin.  While 
the  keeping  quality  of  milk  may  be  materially  increased  by  the  use  of 
such  chemicals,  their  use  has  been  opposed  by  health  authorities  and  is 
contrary  to  the  Pure  Food  Laws.  If  milk  is  handled  with  any  degree 
of  care,  there  should  be  no  need  for  the  use  of  chemical  preservatives. 
They  are  simply  a  means  of  counteracting  the  unsanitary  conditions  of 
the  production  and  handling.  The  same  results  can  be  obtained  by 
cleanliness  in  the  production  of  the  milk  and  the  use  of  low  temperatures 
for  preventing  the  contamination  and  subsequent  growth  of  the 
bacteria  in  the  milk.  The  developments  in  the  production  of  clean 
milk  of  the  past  few  years  have  illustrated  very  clearly  that  the  use  of 
chemical  preservatives  is  not  necessary. 

NORMAL  DEVELOPMENT  OF  MICROORGANISMS  IN  MILK 
The  flora  of  any  particular  sample  of  fresh  milk  is  determined  by  the 
conditions  under  which  it   is  produced.     In   stables   where  extreme 
cleanliness  is  practised  the  flora  may  be  practically  limited  to  those 


THE  RELATION  OF  MICROORGANISMS  TO  MILK 


461 


species  which  occur  in  the  udder  of  the  cows,  but  under  ordinary  condi- 
tions there  will  be  in  addition  to  the  normal  udder  types  such  others  as 
may  occur  on  the  cow's  body  and  in  the  dust  and  atmosphere  of  the 
stables.  Market  milk,  therefore,  when  first  obtained  from  the  cow 
ordinarily  contains  a  mixed  flora,  the  different  types  present  depending 
upon  the  sanitary  conditions  under  which  the  milk  is  produced. 

The  future  development  of  this  initial  flora  is  largely  dependent 
upon  the  temperature  at  which  the  milk  is  kept.  If  the  milk  is  held  at 
temperatures  between  10°  and  21°  there  will  result  what  may  be  con- 
sidered as  the  normal  development  of  milk  fermentations.  These 
changes  may  be  divided  for  convenience  into  four  periods  or  stages. 

FIRST  STAGE.  GERMICIDAL  PERIOD. — It  has  been  shown  by  a  num- 
ber of  investigators  that  instead  of  an  increase  in  the  numbers  of  bacteria 
in  fresh  milk  there  is  normally  a  decrease  in  the  number  during  the 
first  few  hours  after  its  production.  The  rapidity  of  this  decrease  and 
the  length  of  time  over  which  it  extends  seem  to  be  determined  largely 
by  the  temperature  at  which  the  milk  is  kept.  The  higher  the  tempera- 
ture the  more  rapidly  the  number  of  organisms  decreases  and  the  more 
quickly  the  end  of  the  germicidal  period  is  reached.  If  the  tempera- 
tures are  kept  fairly  low  the  rate  of  decrease  is  much  slower  but  the  de- 
cline will  extend  over  a  considerably  longer  period.  This  is  shown  by 
the  following  examples  given  by  Hunziker. 

TABLE  SHOWING  THE  GERMICIDAL  ACTION  IN  Cow's  MILK 


Name 
of 
cow 

Milk, 
warm 
and 
fresh 

Temp.  * 
of 
milk 

After 
hours 

After 
6 
hours 

After 
hours 

After 

12 

hours 

After 
IS 
hours 

After 
hours 

After 
hours 

After 
.48 

hours 

[ 

40° 

i,  080 

1,220 

1,040 

1,  020 

1,120 

i,36o 

1,040 

400 

May  

1,212    -S 

55° 

1,260 

I,4OO 

1,500 

i  462 

I  360 

I 

70° 

1,000 

1,340 

1,  860 

3,46o 

3,460 

64,000 

800,000 

[ 

40° 

4,400 

4,260 

3,620 

3,700 

3,900 

4,000 

3,900 

3,840 

Ida 

5  I2O    T 

55° 

3  900 

•>  460 

2  980 

2  800 

3,240 

I 

70° 

3,560 

2,120 

1,880 

1,880 

1,240 

4,96o 

58,400 

( 

40° 

1,170 

1,070 

1,120 

870 

1,120 

990 

1,  060 

1,080 

Julia  

1.345   1 

55° 

1.080 

990 

980 

1,400 

1,  080 

i,  080 

3.HO 

68,800 

I 

70° 

1.  000 

1,000 

1,200 

5,600 

17,720 

1,600,000 

The  exact  reason  for  this  decline  is  at  present  not  well  understood. 
Some  investigators  believe  that  milk  possesses  a  certain  germicidal  ac- 
tion or  property  which  results  in  the  destruction  of  a  portion  of  the 
organisms  found  in  the  milk  at  the  outset. 

*  Fahrenheit. 


462 


MICROBIOLOGY   OF   MILK   AND   MILK   PRODUCTS 


The  work  of  other  investigators  seems  to  show  that  the  so-called 
germicidal  action  is  felt  by  certain  species  and  not  by  others  as  is  indi- 
cated by  the  following  sample. 


Age  of  milk 

Total 
bacteria 

Acid 
bacteria 

Per  cent,  acid 
bacteria 

Liquefying 
bacteria 

Fresh                 .  .    . 

12.  "^O 

1,  2^o 

IO 

200 

3  hours  

12,250 

2,ooo 

16 

2OO 

6  hours 

10  6^O 

2.2^o 

22 

800 

9  hours  

^6,000 

2O,2^O 

36 

cro 

12  hours 

114  2ZO 

68  400 

60 

I  OOO 

This  would  seem  to  indicate  that  the  decrease  in  number  is  due  not  so 
much  to  a  definite  germicidal  property  possessed  by  the  milk  as  to  the 
gradual  dying  out  of  certain  species  which  for  some  reason  do  not  find 
the  milk  a  suitable  environment  for  development,  while  other  types, 
finding  the  milk  suitable  to  their  needs,  develop  uniformly  from  the 
start. 

Rosenau  and  McCoy  found  that  the  germicidal  properties  of  milk 
were  destroyed  by  boiling  or  by  heating  it  above  80°  and  that  lower 
temperatures  destroyed  it  for  certain  organisms.  These  workers  also 
found  that  there  was  marked  agglutination  of  the  organisms  in  raw  milk 
and  conclude  that  this  accounts  for  the  decreased  number  of  colonies 
developing  in  plate  cultures  and  that  the  germicidal  action  is  therefore 
more  apparent  than  real. 

SECOND  STAGE.  PERIOD  FROM  END  OF  GERMICIDAL  ACTION  TO 
TIME  OF  CURDLING. — The  period  following  immediately  after  the  ger- 
micidal action  is  characterized  by  the  rapid  development  of  the  lactic 
organisms.  Under  normal  conditions  this  group  develops  much  more 
rapidly  than  any  other  type.  Not  only  do  they  increase  rapidly  in 
actual  numbers  but  their  percentage  also  rises  rapidly.  There  may 
be  a  continual  increase  in  numbers  in  the  other  species,  but  their  growth 
is  much  less  rapid  than  that  of  the  Bad.  lactis  acidi  type.  As  this  period 
advances  certain  of  the  miscellaneous  types  may  cease  to  grow  entirely. 
During  this  time  the  gas-producing  acid  organisms  of  the  B.  coli  and 
Bact.  lactis  aerogenes  type  may  develop  more  or  less  rapidly,  but  if  the 
milk  is  held  at  temperatures  not  much  above  20°,  the  Bact.  lactis  acidi 
type  will  develop  much  more  rapidly,  so  that  by  the  time  the  milk  be- 
comes sour  and  curdles,  this  type  will  constitute  99  per  cent  approxi- 


THE   RELATION   OF   MICROORGANISMS   TO   MILK  463 

mately  of  the  total  number  in  the  milk.  From  the  standpoint  of  the 
milk  consumer  milk  ceases  to  be  of  value  when  the  end  of  this  period  is 
reached,  but  there  are  further  developments  which  are  of  importance  in 
certain  lines  of  dairy  manufactures,  notably  cheese  making. 

THIRD  STAGE.  PERIOD  FROM  TIME  OF  CURDLING  UNTIL  ACIDITY 
is  NEUTRALIZED. — At  the  time  milk  curdles  it  contains  enormous  num- 
bers of  the  lactic  bacteria.  The  number  usually  runs  into  the  millions 
and  may  be  even  higher  than  one  thousand  million  per  c.c.  By  the 
time  the  coagulation  takes  place  the  acidity  of  the  milk  is  so  high  that 
the  growth  of  the  lactic  organisms  is  checked  and  from  this  time  on 
their  number  decreases  with  more  or  less  rapidity. 

During  the  period  following  the  curdling  certain  other  types  of 
organisms  which  have  remained  more  or  less  dormant  in  the  milk  during 
the  earlier  stages  now  begin  to  grow.  The  organisms  especially  impor- 
tant in  this  stage  are  Oidium  lactis,  certain  species  of  molds,  and  yeasts. 
These  organisms  are  able  to  grow  in  a  highly  acid  medium,  and  as  a 
result  of  their  development  the  acid  is  decreased  until  the  milk  finally 
presents  a  neutral  or  alkaline  condition  resulting  from  the  decomposi- 
tion of  the  proteins  in  the  milk. 

FOURTH  STAGE.  FINAL  DECOMPOSITION  CHANGES. — The  reduction 
of  the  acidity  affords  favorable  conditions  for  the  growth  of  certain  types 
of  organisms  which  have  remained  in  the  milk  during  the  earlier  stages 
but  have  been  practically  dormant.  In  this  fourth  stage  the  conditions 
are  suitable  for  the  growth  of  the  liquefying  and  peptonizing  bacteria 
and  they  now  grow  rapidly,  causing  the  decomposition  of  the  casein. 
The  changes  resulting  from  this  type  of  organisms  are  of  special  signifi- 
cance in  cheese  making  and  are  discussed  more  fully  in  another  chapter. 

ABNORMAL  FERMENTATIONS  IN  MILK 

GASSY  FERMENTATION.— It  frequently  happens  that  instead  of  the 
normally  rapid  development  of  the  Bact.  lactis  acidi  type  of  organisms 
in  the  milk,  other  acid  producers  develop  rapidly,  with  the  production 
of  more  or  less  gas.  The  organisms  most  prominent  in  this  type  of  fer- 
mentation are  the  B.  coli  communis  and  the  Bact.  lactis  aerogenes  types. 
This  group  of  organisms  contains  a  number  of  varieties,  some  of  which 
produce  little  or  no  gas  while  others  develop  large  amounts.  Their 
action  in  milk  is  usually  accompanied  by  disagreeable  odors  and  flavors. 
They  grow  readily  in  the  presence  of  air  and  therefore  develop  abundant 
colonies  on  the  surface  of  plate  cultures.  This  distinguishes  the  mem- 


464 


MICROBIOLOGY   OF   MILK  AND   MILK  PRODUCTS 


bers  of  this  group  quite  clearly  from  those  of  the  true  lactic  group  which 
grow  chiefly  below  the  surface  of  the  medium.  The  members  of  this 
group  do  not  form  spores,  but  certain  varieties  are  quite  resistant  to 
heat  and  will  oft  times  survive  pasteurizing  temperatures  which  com- 
pletely destroy  the  Bact.  lactis  acidi  group.  They  grow  most  rapidly 
at  high  temperatures,  between  20°  and  37°. 

SWEET  CURDLING  FERMENTATION. — This  phenomenon  is  caused  by 
a  variety  of  organisms  which  cause  the  milk  to  coagulate  without  the 
production  of  acid.  The  coagulation  is  brought  about  by  a  rennet-like 
enzyme  produced  by  this  type  of  bacteria.  The  resulting  milk  is 
either  neutral  or  alkaline  in  reaction.  Usually  the  coagulation  of  the 
milk  is  followed  by  the  digestion  of  the  casein  as  a  result  of  another 
enzyme  which  is  also  produced  by  these  bacteria.  The  coagulation 
caused  by  these  organisms  is  slower  than  in  the  case  of  the  acid 
formers  and  the  curd  is  usually  soft  and  mushy  as  compared  with  the 
curd  formed  in  the  normal  acid  fermentation.  The  members  of  this 
group  get  into  the  milk  from  and  along  with  dust 
and  dirt  associated  with  unsanitary  conditions. 
Some  of  the  species  produce  spores  and  are  not 
killed  by  the  ordinary  methods  of  pasteurization. 
This  fact  accounts  for  the  occurrence  of  sweet 
curdling  of  pasteurized  milk.  This  group ^of  or- 
ganisms is  unable  to  develop  rapidly  in  the  pres- 
ence of  the  lactic  bacteria  and  for  this  reason  we 
do  not  commonly  get  the  sweet  curdling  of  raw 
milk.  The  presence  of  these  organisms  is  evi- 
dence of  insanitary  conditions.  Frequently  they 
develop  very  disagreeable  flavors  in  the  milk. 

ROPY  OR  SLIMY  FERMENTATION. — One  of  the 
most  common  milk  infections  causing  trouble  to 
the  milk  dealer  is  that  which  causes  a  ropy  or 
slimy  fermentation  of  milk.  This  is  sometimes  spoken  of  as  stringy 
milk  (Fig.  143).  Several  species  of  organisms  are  capable  of  pro- 
ducing this  condition.  These  organisms  grow  most  freely  in  the 
presence  of  an  abundant  supply  of  oxygen  and  for  this  reason  the  cream 
usually  becomes  slimy  before  any  changes  are  apparent  in  the  under- 
lying layers  of  milk.  B.  lactis  viscosus  is  perhaps  the  most  common 
species  in  this  group.  The  slimy  condition  in  the  milk  is  supposed 


FIG.  143.  — Ropy 
cream  lifted  with  a  fork. 
(After  Ward.) 


THE    RELATION    OF   MICROORGANISMS    TO   MILK 


465 


to  be  the  result  of  a  very  viscid  capsule  surrounding  these  organisms 
(Fig.  144).  Representatives  of  this  group  are  quite  resistant  to  heat 
and  frequently  pass  uninjured  through  the  methods  of  cleansing  and 
scalding  used  under  ordinary  dairy  conditions.  Because  of  this, 
dairy  utensils  once  infected  may  become  a  constant  source  of  infection. 


FIG.  144. — Bacillus  lactis  viscosus  from  a  milk  culture.     (After  Ward.} 

Ihis  trouble  can  be  effectively  stopped  by  a  thorough  scalding  of  all 
utensils  coming  in  contact  with  the  milk. 

BITTER  FERMENTATION. — Bitter  flavors  in  milk  may  be  the  result 
of  bacterial  changes  after  the  milk  has  been  drawn,  or  due  to  certain 
feeds  which  the  cows  have  consumed.  If  the  cows  are  allowed 
to  eat  certain  kinds  of  vegetation,  such  as  "rag  weed"  and  certain 
other  plants,  they  may  impart  a  bitter  taste  to  the  milk,  in  which  case 
the  abnormal  flavor  will  be  apparent  when  the  milk  is  fresh  and  usually 
becomes  less  pronounced  as  the  milk  becomes  older,  because  of  the 
volatile  nature  of  the  substances  causing  the  bitterness.  Most  of  the 
cases  of  bitter  milk  and  cream,  however,  are  due  to  the  growth  of 
certain  types  of  bacteria  in  which  case  the  bitterness  increases  in  in- 
tensity with  the  age  of  the  milk.  Some  of  the  species  capable  of 
producing  bitter  milk  grow  at  quite  low  temperatures,  which  accounts 
for  the  fact  that  the  most  trouble  with  bitter  flavors  is  found  in  milk 
and  cream  which  has  been  held  at  low  temperatures  for  some  time. 

30 


466 


MICROBIOLOGY   OF   MILK   AND  MILK   PRODUCTS 


ALCOHOLIC  FERMENTATION. — The  bacteria  as  a  group  are  not 
able  to  act  on  the  milk  sugar  and  produce  alcohol,  but  it  sometimes 
happens  that  yeasts  get  into  the  milk  in  sufficient  numbers  to  ferment 
the  milk  sugar,  producing  appreciable  amounts  of  alcohol.  To  the 
milk  handler  this  trouble  is  not  usually  serious  but  the  action  of  the 
yeasts  is  frequently  of  considerable  importance  in  the  cheese  industry. 

OTHER  FERMENTATIONS. — It  frequently  happens  that  a  consider- 
able variety  of  disagreeable  flavors  and  odors  develop  in  milk.  These 
may  be  due  to  the  direct  absorption  of  odors  from  the  foul  stable 
atmosphere  or  strong-smelling  feeds,  such  as  silage;  or  they  may  be, 
and  no  doubt  frequently  are,  the  result  of  the  growth  of  certain  types 
of  bacteria  which  have  entered  the  milk  from  dirty  surroundings. 
The  growth  of  some  of  these  organisms  is  frequently  the  cause  of  the 
so-called  cowy  and  stable  odors  and  flavors. 

COMMERCIAL  SIGNIFICANCE  OF  MICROORGANISMS  IN  MILK 
RELATION  OF  DIRT  CONTAMINATION  TO  GERM  CONTENT. — To  the 
commercial  milkman  bacteria  are  of  importance  only  as  they  influence 
the  length  of  time  the,  milk  will  keep  in  a  salable  condition.  The 
consumers  do  not  want  milk  that  is  sour  or  has  unpleasant  flavors 
and  odors.  In  order  to  sell  his  milk,  therefore,  the  milkman  must 
avoid  the  presence  of  these  undesirable  conditions,  and  in  proportion 
as  he  recognizes  the  relation  between  germ  life  and  the  quality  of 
his  product,  will  he  pay  attention  to  the  presence  and  development 
of  microorganisms  in  his  milk.  In  like  manner,  the  presence  or  ab- 
sence of  dirt  contamination  is  important  from  the  commercial  stand- 
point since  it  bears  a  relation  to  the  bacterial  count,  and,  therefore, 
affects  the  keeping  properties  of  the  milk.  Under  normal  conditions 
there  is  .a  fairly  direct  relation  between  the  amount  of  visible  or  soluble 
dirt  and  the  number  of  bacteria  found  in  any  given  lot  of  fresh  milk. 
This  relation  may  be  shown  by  the  following  samples  taken  from  four 
different  milk  producers: 


Producer 

Number  of 
samples 

Average  mg. 
dry  dirt  per  liter 

Average  number 
bacteria  per 
c.c. 

Average  hours 
to  time  of  curdling 

A 

5 

ci  .  c 

115,000 

175 

B  . 

16 

58.8 

273,600 

78 

c  

21 

70.0 

428,600 

75 

D  

17 

7I.Q 

949,400 

68 

THE   RELATION   OF  MICROORGANISMS   TO   MILK  467 

This  relation  does  not  always  hold  for  the  reason  that  a  gram  of 
one  kind  of  dirt  may  contain  infinitely  more  organisms  than  an  equal 
amount  of  some  other  kind.  The  difference  in  the  solubility  of  various 
forms  of  dirt  always  causes  apparent  discrepancies  in  this  normal 
relation.  In  the  majority  of  cases,  however,  the  relation  shown  in 
the  above  examples  will  hold  reasonably  true  in  the  case  of  fresh  milk. 
There  is  also  a  general  relation  between  the  number  of  bacteria  in 
fresh  milk  and  the  length  of  time  it  will  keep  before  souring  and  curd- 
ling. In  this  case  the  relation  is  in  inverse  ratio,  the  smaller  the  initial 
contamination,  the  longer  the  keeping  time,  and  vice  versa.  This  re- 
lation is  also  shown  in  the  table  given  above.  There  are  many  ir- 
regularities, however,  in  this  relation  because  of  differences  in  the 
flora  of  fresh  milk.  It  may  frequently  happen  that  a  sample  of  milk 
containing  a  relatively  high  number  of  organisms  will  not  sour  as 
quickly  as  another  sample  with  a  smaller  original  germ  content.  The 
associative  action  of  the  different  species  of  organisms  is  an  important 
factor  here.  In  making  comparisons  of  this  sort,  it  is,  of  course, 
necessary  that  the  different  samples  be  held  at  the  same  temperatures. 

MILK  AS  A  CARRIER  OF  DISEASE-PRODUCING  ORGANISMS 

It  is  not  the  purpose  of  this  chapter  to  discuss  in  detail  the  diseases 
which  may  be  carried  by  milk,  but  a  chapter  on  bacteriology  of  milk 
would  be  incomplete  without  a  brief  discussion  of  this  important 
subject. 

From  the  standpoint  of  their  relation  to  the  health  of  the  con- 
sumer the  microorganisms  in  milk  may  be  divided  into  three  groups 
on  the  basis  of  whether  they  are  beneficial,  inert  or  injurious  to  health. 

Acid  Forms. — The  preservative  properties  of  sour  milk  have  been 
known  since  very  ancient  times.  Its  use  as  a  preservative  for  meat, 
eggs  and  other  perishable  food  products  demonstrates  the  value  of 
sour  milk  as  a  means  of  preventing  decomposition.  It  has  also  been 
known  for  a  long  time  that  sour  milk  has  a  certain  therapeutic  value 
because  of  the  action  of  the  lactic  bacteria  in  preventing  harmful 
fermentations  in  the  digestive  tract.  More  recently  the  work  of 
Metchnikoff  has  shown  the  usefulness  of  sour  milk  both  for  the  treat- 
ment and  prevention  of  intestinal  disorders  by  inhibiting  the  develop- 
ment of  the  putrefactive  bacteria  in  the  digestive  tract.  In  view  of 
the  value  of  sour  milk  for  preventing  certain  forms  of  disease  and  its 


468  MICROBIOLOGY   OF   MILK   AND    MILK    PRODUCTS 

inhibiting  action  on  certain  undesirable  organisms  the  Bact.  lactis 
acidi  type  of  bacteria  must  be  regarded  as  beneficial  organisms,  and 
from  the  standpoint  of  the  health  of  the  consumer  their  presence  in  the 
milk  is  to  be  welcomed  rather  than  discouraged.  As  the  value  of 
sour  milk  drinks  becomes  better  known  the  importance  of  this  group 
of  milk  bacteria  will  be  more  fully  recognized. 

Neutral  or  Inert  Forms. — In  ordinary  milk  there  is  a  large  class  of 
bacteria  which,  so  far  as  known,  have  no  appreciable  effect  either 
upon  the  composition  of  the  milk  or  the  health  of  the  persons  consuming 
it.  This  group  includes  a  number  of  species,  many  of  them  being 
coccus  forms,  some  of  them  appearing  in  plate  cultures  as  chromogenic 
colonies.  They  grow  more  or  less  freely  in  milk,  depending  upon  the 
conditions,  but  they  are  usually  held  in  check  by  the  acid-forming  bac- 
teria and  do  not  constitute  a  very  important  part  of  the  flora  of  normal 
milk.  They  are,  therefore,  of  little  significance  from  the  practical 
standpoint  except  as  they  indicate  the  conditions  under  which  the  milk 
has  been  produced  and  handled. 

Injurious  Organisms. — The  diseases  which  may  be  carried  by  milk 
are  of  two  classes. 

Epidemic  Diseases. — The  human  diseases  most  commonly  carried 
by  milk  are  typhoid  fever,  diphtheria  and  scarlet  fever  and  occasionally 
other  diseases  such  as  septic  sore-throat,  cholera  and  foot-and-mouth 
disease.  The  first  three  are  by  far  the  most  important  of  this  group. 
The  outbreaks  of  typhoid  fever  which  are  traceable  to  milk  occur 
most  frequently.  There  is  a  large  accumulation  of  data  showing  the 
occurrence  of  epidemics  caused  by  infected  milk.  An  epidemic  caused 
by  the  milk  supply  has  certain  characteristics  which  distinguish  it 
from  epidemics  resulting  from  other  causes.  A  considerable  number 
of  cases  of  the  particular  disease  will  appear  almost  simultaneously 
and  will  be  distributed  along  some  particular  milk  route.  Usually 
the  epidemic  stops  as  suddenly  as  it  began  except  for  a  few  secondary 
cases  contracted  from  those  first  taken.  The  source  of  the  disease 
organisms  is  a  human  patient  suffering  from  the  disease.  The  infection 
of  the  milk  may  be  direct,  as  when  a  sick  person  handles  a  milk, 
or  it  may  be  indirect  as  when  a  person  caring  for  a  patient  also  works 
about  the  milk.  In  other  cases  it  may  be  caused  by  contamination 
of  the  water  used  in  washing  the  utensils  or  by  cows  wading  in  water 
of  infected  streams  and  getting  the  organisms  on  their  body  whence 


THE   RELATION   OF   MICROORGANISMS   TO   MILK 


469 


they  fall  into  the  milk  pail  at  milking  time.     The  return  of  milk  bottles 
from  the  sick  room  sometimes  is  the  means  of  infecting  the  milk  supply. 


FIG.  145. 

Unfortunately  the  specific  organisms  of  these  diseases  grow  readily 
in  milk  and  a  small  infection  is  all  that  is  necessary  to  render  the 
milk  dangerous  by  the  time  it  reaches  the  consumer. 


470  MICROBIOLOGY    OF   MILK    AND    MILK   PRODUCTS 

Non-epidemic  Diseases. — There  is  another  class  of  diseases  which 
may  be  carried  by  milk  which  are  not  characterized  by  a  sudden  out- 
break, and  for  this  reason  are  not  so  readily  recognized  as  being  asso- 
ciated with  the  milk  supply.  One  of  these  diseases,  namely  tuber- 
culosis, is  caused  by  the  specific,  well-known  organism,  Bad.  tuberculo- 
sis, which  may  get  into  the  milk  from  the  udder  of  a  tuberculous  cow 
or  by  the  organisms  which  have  been  given  off  from  the  digestive 
tract  of  the  animal  becoming  scattered  about  the  stable  and  finally 
getting  into  the  milk  with  particles  of  dust  and  filth.  In  some 
cases  the  milk  may  become  infected  by  persons  having  the  disease 
being  permitted  to  handle  the  milk.  Fortunately  for  mankind  Bact. 
tuberculosis  does  not  multiply  in  milk. 

Regarding  the  danger  of  contracting  tuberculosis  from  the  use  of 
milk  there  is  at  present  some  difference  of  opinion,  but  the  con- 
sensus of  opinion  at  the  present  time  seems  to  be  that  there  may 
not  be  very  great  danger  for  healthy  adults,  but  that  a  considerable 
percentage  of  the  cases  of  tuberculosis  of  children  may  be  traced  from 
the  milk  supply.  Fortunately  none  of  these  specific  disease  organisms 
produce  spores  and  the  temperatures  used  in  the  process  of  pasteuriza- 
tion by  the  "holding"  method  are  sufficient  to  destroy  any  of  the  dis- 
ease bacteria  known  to  be  carried  by  milk. 

There  is  another  class  of  disorders  not  so  well  defined  as  the  above 
but  which  are  nevertheless  of  great  importance  from  the  standpoint  of 
public  health,  especially  of  young  children  and  also  to  some  extent  of 
adults.  This  group  includes  such  disorders  as  infantile  diarrhoea,  sum- 
mer complaint,  cholera  infantum  and  other  disorders  of  the  digestive 
tract.  The  organisms  producing  these  troubles  doubtless  belong  to  the 
group  of  putrefactive  bacteria  which  come  from  filth.  Some  of  the  gas 
producers  and  some  of  the  peptonizers  are  probably  responsible  for 
these  troubles.  Shiga  isolated  from  a  large  number  of  cases  of  infant 
diarrhoea  a  bacterium  which  he  named  Bact.  dysenteries,  but  in  general 
the  specific  organisms  responsible  for  these  intestinal  troubles  are  not 
well  known.  Their  importance,  however,  is  shown  by  the  relation  of 
the  germ  content  of  milk  to  infant  mortality  (see  Fig.  145). 
BACTERIOLOGICAL  ANALYSIS  OF  MILK 

The  development  of  our  knowledge  of  the  relation  of  bacteria  to  the 
wholesomeness  of  foods  has  led  to  a  study  of  the  bacterial  content  of 
milk  as  a  means  of  determining  its  purity.  The  methods  used  for  this 
purpose  have  followed  quite  closely  those  of  the  water  bacteriologists. 


THE   RELATION   OF   MICROORGANISMS    TO   MILK  471 

For  many  years,  dairy  bacteriologists  have  endeavored  to  determine  the  number 
of  organisms  in  milk  by  plating  it  into  nutrient  agar  or  gelatin.  By  this  method 
the  number  of  colonies  developing  in  the  plates  is  assumed  to  represent  the  germ 
content  of  the  milk.  But  even  when  the  best  methods  are  employed,  the  plate  count 
represents  only  the  approximate  and  not  the  exact  number  of  bacteria  in  any  lot  of 
milk.  It  should  also  be  borne  in  mind  that  such  counts  are  always  underestimates, 
because  of  the  fact  that  not  all  species  will  develop  in  any  given  medium  or  incubation 
temperature.  The  careful  worker  can  recognize  certain  types  of  bacteria  in  plain 
media,  but  the  addition  of  blue  litmus  solution  to  either  agar  or  gelatin,  greatly 
assists  in  the  differentiation  of  types  and  species. 

THE  DIRECT  MICROSCOPIC  METHOD. — The  plating  method  is  expensive  because 
of  the  large  amount  of  time  and  materials  needed.  It  is  not  possible  for  one  person 
to  handle  a  large  number  of  samples  at  one  time.  In  routine  work  in  the  city  labora- 
tories this  labor  has  been  a  serious  drawback  to  this  method.  In  order  to  decrease 
the  labor  and  give  greater  possibilities  to  the  work  Stewart  devised  a  method  by 
which  the  bacterial  condition  of  milk  can  be  studied  by  direct  microscopic  examina- 
tion. His  purpose  was  to  determine  only  the  species  present,  but  later  Slack  and 
still  more  recently  Breed  developed  the  method  for  determining  the  approximate 
numbers  as  well  as  the  general  species  present  in  a  given  sample  of  milk. 

LEUCOCYTES. — The  microscopic  examination  of  milk  sediment  revealed  the  fact 
that  frequently  a  sample  would  be  found  which  showed  the  presence  of  leucocytes  in 
greater  or  less  numbers.  The  presence  of  these  cells  was  regarded  as  important 
because  it  was  assumed  that  they  showed  the  presence  of  inflammation  and  pus  for- 
mation in  the  udders  of  the  cows  producing  the  milk. 

Several  methods  have  been  used  for  determining  the  leucocyte  content  of  milk. 
"The  Smear  Sediment"  and  "Blood  Counter"  are  methods  which  more  strictly 
belong  to  laboratory  practices  and  will  not  be  considered  in  this  place. 

BACTERIOLOGICAL  MILK  STANDARDS 

The  relation  of  the  bacterial  content  of  milk  to  its  wholesomeness 
has  led  to  the  adoption  of  certain  standards  by  the  boards  of  health  in 
our  cities.  These  standards  recognize  the  fact 'that  the  germ  content  of 
milk  in  the  large  cities  is  greater  than  in  the  smaller  ones  because  of  the 
greater  distance  from  which  it  is  shipped  and  its  age  on  arrival  to  the 
city.  New  York  City  in  1900  adopted  a  maximum  limit  of  1,000,000 
per  c.c.  Later  Boston  established  a  limit  of  500,000,  Chicago  1,000,000 
from  May  to  September,  inclusive,  and  500,000  from  October  to  April, 
inclusive  and  Rochester  100,000.  Other  cities  have  made  similar 
standards. 

Stokes'  standard  for  the  number  of  leucocytes  permissible  in  normal 
milk  was  5  per  field  of  the  Jf  2  objective  in  his  smeared  sediment  prepa- 
ration. Bergey  found  so  many  samples  running  above  this  number 
that  he  made  the  limit  10  cells  per  field  and  felt  that  no  milk  containing 


472  MICROBIOLOGY   OF  MILK   AND   MILK  PRODUCTS 

more  than  this  number  should  be  used  for  food.  Later  Slack  raised 
the  limit  to  50  cells  per  field.  The  reason  for  changing  the  stand- 
ard was  due  partly  to  the  larger  numbers  found  as  a  result  of  improved 
methods  but  more  especially  to  the  discovery  that  milk  from  appar- 
ently healthy  cows  normally  contains  leucocytes  in  excess  of  the  first 
standards  set. 

With  the  development  of  the  dairy  score  card,  there  was  a  decided 
tendency  to  place  emphasis  on  the  sanitary  conditions  at  the  farm  rather 
than  on  the  germ  content  of  the  milk.  But  it  was  soon  discovered  that 
the  farm  score  did  not  necessarily  show  the  true  condition  of  the  milk, 
and  at  present,  the  tendency  seems  to  be  toward  placing  more  confidence 
in  the  germ  content  as  the  best  measure  of  the  true  conditions  of  pro- 
duction and  handling.  However,  the  fact  must  be  recognized  that  our 
methods  of  bacteriological  analysis  are  not  sufficiently  accurate  to 
justify  the  bacteriologist  in  passing  judgment  concerning  the  quality  of 
any  milk  supply  on  a  single  analysis.  In  order  to  secure  results  which 
are  at  all  trustworthy,  a  series  of  analyses  must  be  considered. 

It  is  held  by  some  that  a  numerical  standard  is  of  little  value  since 
the  actual  number  of  organisms  present  in  a  given  lot  of  milk  may  not  be 
a  correct  measure  of  its  wholesomeness.  For  this  reason  some  cities 
pay  little  attention  to  the  numbers  of  bacteria  present  but  base  their 
standards  wholly  on  the  species  and  the  quality  of  the  milk  is  judged  on 
the  presence  and  numbers  of  streptococci,  B.  coli,  leucocytes,  sediment. 
Milk  is  passed  or  condemned  on  the  basis  of  any  one  or  combination  of 
these  conditions. 

In  recent  years  there  has  been  a  tendency  to  combine  these  two 
standards  using  the  total  germ  content  as  a  measure  of  the  care  the 
milk  has  had  and  the  presence  or  absence  of  certain  groups  or  species  as 
an  indication  of  the  occurrence  of  pathological  conditions  in  the  cows 
producing  the  milk.  The  practice  in  most  city  laboratories  now  is  to 
make  use  of  both  the  numbers  and  the  species  present  in  determin- 
ing the  quality  of  the  milk  supply. 

VALUE  or  BACTERIOLOGICAL  MILK  STANDARDS  AND  ANALYSES 
Regarding  the  value  of  bacteriological  standards  for  milk  there  is 
still  some  difference  of  opinion  among  milk  bacteriologists.  The  germ 
content  of  any  lot  of  milk  is  largely  dependent  upon  three  factors:  the 
number  of  organisms  getting  into  the  fresh  milk;  the  temperature  at 
which  it  is  kept;  the  age  of  the  milk  when  analysis  is  made. 


THE   RELATION   OF   MICROORGANISMS   TO   MILK  473 

The  high  bacterial  count  in  any  lot  of  milk  may  be  the  result  of  any 
one  of  these  conditions  or  a  combination  of  them.  A  high  count  means 
that  there  has  been  carelessness  either  in  the  production,  resulting  in 
high  initial  contamination,  or  in  the  subsequent  handling  permitting  a 
rapid  multiplication  of  the  organisms,  or  that  the  milk  is  old. 

On  the  other  hand,  milk  with  a  low  germ  content  can  be  obtained 
only  where  the  original  contamination  is  small  and  the  milk  has  been 
held  at  low  temperatures.  A  low  count,  therefore,  means  care  both  in 
the  production  and  later  handling  of  the  milk. 

While  the  germ  content  may  be  regarded  as  a  general  index  to  the 
care  the  milk  has  received,  it  may  not  at  all  indicate  its  wholesomeness. 
A  high  count  may  be  the  result  of  the  rapid  growth  of  the  lactic  bacteria, 
in  which  case  the  milk  may  be  perfectly  safe  and  wholesome.  On  the 
other  hand,  the  count  maybe  quite  small  but  contain  pathogenic  species. 
The  bacteria  count  is  valuable  as  showing  the  sanitary  conditions  of 
production  and  handling,  but  much  care  should  be  used  in  the  inter- 
pretation of  such  results.  In  some  ways  a  direct  microscopic  examina- 
tion of  the  milk  sediment  is  much  more  satisfactory.  The  skilled 
analyst  can  recognize  certain  types  which  may  indicate  the  sanitary 
quality  of  the  milk.  With  sufficient  experience  one  can  recognize  strep- 
tococci, certain  other  groups  and  leucocytes.  The  presence  and  abun- 
dance of  one  or  more  of  these  groups  may  indicate  the  nature  of  the 
original  contamination  and  the  existence  of  diseases  in  the  udders  of 
cows.  If  rightly  interpreted  the  information  thus  obtained  is  of  much 
value.  The  weakness  of  this  method  lies  in  the  fact  that  it  is  not 
possible  to  recognize  all  types  of  disease  organisms.  In  a  smear  prepa- 
ration it  is  not  possible  to  differentiate  between  pathogenic  and  non- 
pathogenic  streptococci  or  between  B.  coli  and  certain  other  types. 
The  presence  of  unusual  numbers  of  streptococci  and  pus  cells  may 
indicate  the  existence  of  disease  in  the  cows  and  when  this  condition  is 
found  in  the  milk  it  is  often  possible  to  trace  it  back  to  the  farm,  lo- 
cate the  diseased  cow  and  prevent  her  milk  from  being  used  for  human 
consumption. 

The  tendency  at  present  is  to  combine  the  quantitative  and  quali- 
tative analyses  and  the  results  thus  obtained  in  the  hands  of  the  careful 
worker  are  of  much  practical  value  in  controlling  the  quality  of  a  city's 
milk  supply. 


CHAPTER  II* 
THE  RELATION  OF  MICROORGANISMS  TO  BUTTER 

Butter  is  the  fat  of  milk  that  has  been  largely  freed  from  the  other 
constituents  of  milk  by  the  processes  of  creaming  and  churning. 
If  milk  is  allowed  to  stand,  the  fat,  which  is  in  the  form  of  minute 
globules,  accumulates  in  the  upper  layers  of  the  milk  because  its  spe- 
cific gravity  is  much  lower  than  that  of  milk  serum.  In  modern  prac- 
tice the  fat  is  concentrated  in  a  portion  of  the  milk  by  passing  the  milk 
through  a  cream  separator.  In  the  rapidly  revolving  bowl  of  the  separa- 
tor the  centrifugal  force  exerted  is  many  times  greater  than  that  of  grav- 
ity and  the  fat  is  rapidly  and  efficiently  removed.  The  cream,  which  is 
obtained  by  these  methods,  contains  varying  amounts  of  fat  which 
is  further  concentrated,  by  subjecting  it  to  agitation  in  the  churning 
process.  The  globules  of  fat  cohere  to  form  larger  and  larger  masses 
until  the  entire  amount  of  fat  is  brought  into  a  single  mass,  the  butter. 

TYPES  or  BUTTER 

SWEET-CREAM  BUTTER. — If  little  or  no  increase  in  the  acidity  of 
the  milk  or  cream  develops,  previous  to  churning,  the  butter  will  have 
certain  marked  characteristics  and  is  called  sweet  cream  butter.  It 
is  especially  characterized  by  its  low  flavor,  since  it  has  only  the 
flavor  of  the  fat  of  milk  which  is  not  marked.  This  is  usually  known  as 
the  primary  flavor  of  butter.  Sweet-cream  butter  is  also  marked  by 
the  rapidity  with  which  it  undergoes  decomposition  changes,  especially 
when  it  is  made  from  raw  cream. 

SOUR-CREAM  BUTTER. — If  the  cream  is  allowed  to  undergo  the  acid 
fermentation,  the  butter  will  differ  markedly  both  in  degree  and  kind 
of  flavor  from  that  prepared  from  sweet  cream,  and  as  a  rule  its  keeping 
qualities  are  much  better  than  those  of  sweet-cream  butter.  This  type 
of  butter  is  made  throughout  northern  Europe,  England  and  her 

•  Prepared  by  E.  G.  Hastings. 

474 


RELATION   OF   MICROORGANISMS   TO  BUTTER  475 

colonies,  and  in  America.  It  may  be  said  to  be  the  standard  butter 
of  the  world  since  it  is  the  type  made  in  all  the  great  dairy  countries. 
Sweet-cream  butter  is  made  especially  in  southern  Europe,  and  in 
limited  amounts  in  other  countries. 

The  intensity  and  kind  of  flavor  of  butter  is  thus  dependent  on 
the  acid  fermentation  of  the  milk  or  cream.  It  is  not  believed  that 
the  fat  undergoes  any  changes  during  the  acid  fermentation  of  the  milk 
which  could  produce  the  flavor  of  sour-cream  butter,  but  rather  that 
the  increase  in  flavor  is  due  to  the  absorption  by  the  butter  fat  of  certain 
of  the  compounds  formed  in  the  acid  fermentation.  It  is  not  essential 
that  the  fat  be  present  during  the  acid  fermentation  in  order  to  impart 
flavor  to  the  butter.  If  sweet  cream  is  mixed  with  sour  milk  and 
churned  at  once,  the  flavoring  compounds  are  absorbed  by  the  fat  from 
the  fermented  milk,  and  the  butter  will  have  much  the  same  flavor, 
both  as  to  intensity  and  kind,  as  though  the  fat  had  been  present 
during  the  fermentation.  The  churning  of  a  mixture  of  sweet  cream 
and  sour  milk  is  used  commercially  and  is  identical  with  the  methods 
employed  by  the  manufacturers  of  oleomargarine  and  renovated 
butter  to  impart  flavor  to  the  flavorless  fats  they  employ.  It  is 
impossible  to  recognize  these  substitutes  for  butter  by  their  flavor 
since  it  is  identical  with  and  derived  from  the  same  source  as  the  flavor 
of  butter. 

In  the  past  many  ideas  have  been  expressed  as  to  the  source  of 
the  flavor  of  butter;  some  have  asserted  that  it  is  due,  in  part,  to  the 
decomposition  of  the  proteins  of  milk  by  proteolytic  bacteria.  Both 
practical  experience  and  experimental  work  have  demonstrated  the 
connection  between  the  acid  fermentation  of  milk  and  the  flavor  of 
butter,  and  it  is  certain  that  what  is  now  considered  the  finest  type  of 
butter  can  be  made  from  cream  in  which  only  acid-forming  bacteria 
(see  Chap.  I)  have  grown. 

FLAVOR  OF  BUTTER 

CONTROL  OF  BUTTER  FLAVOR. — The  commercial  value  of  any 
sample  of  butter  is  largely  determined  by  its  flavor.  If  it  is  lacking 
in  flavor  and  aroma,  or  if  it  has  a  poor  flavor,  it  brings  a  low  price.  The 
importance  of  being  able  to  control  the  flavor,  both  as  to  degree  and 
kind,  in  the  manufacture  of  butter  has  increased  greatly  in  recent 


476  MICROBIOLOGY   OF   MILK  AND   MILK  PRODUCTS 

years,  because  of  the  introduction  of  the  creamery  system,  which  has 
largely  supplanted  the  making  of  butter  on  the  farm.  The  financial 
success  of  any  creamery  is  largely  dependent  upon  the  ability  of  the 
butter  maker  to  control  the  flavor  of  the  product,  so  that  it  shall  be 
uniform  from  day  to  day.  It  is  asserted  that  one  of  the  factors 
in  the  remarkable  invasion  of  Denmark  into  the  butter  markets  of 
the  world  is  the  uniformity  of  the  Danish  butter,  not  only  from  a  single 
creamery,  but  from  all  the  creameries  of  the  country.  To  the  Danes 
we  owe  the  most  improved  methods  for  the  control  of  the  flavor  of 
butter. 

The  other  points,  texture,  color  and  salt,  which  the  judge  of  butter 
takes  into  consideration,  can  be  easily  controlled,  since  they  are  due  to 
mechanical  operations.  The  flavor,  on  the  other  hand,  is  due  to  the  by- 
products which  are  formed  by  microorganisms  in  the  fermentation  of  the 
milk  and  cream,  and  which  are  absorbed  and  held  by  the  fat.  If  any  of 
the  products  formed  possesses  a  disagreeable  taste  or  an  offensive  odor, 
the  flavor  and  aroma  of  the  butter  will  be  impaired.  It  is  thus  evident 
that  the  control  of  the  flavor  of  butter  is  dependent  on  the  control  of  the 
acid-forming  bacteria  that  ferment  the  milk  and  cream.  This  is  the  prob- 
lem of  the  modern  butter-maker  and  the  modern  methods  seek  to  give 
him  this  control,  to  enable  him  to  eliminate  the  undesirable  bacteria,  B. 
coll  and  Bact.  aero  genes,  the  second  group,*  and  to  insure  the  predomi- 
nance of  the  desirable  bacteria,  Bact.  lactis  acidi.  This  general 
statement  is  not  to  be  interpreted  as  meaning  that  all  bacteria  that 
injure  the  flavor  of  butter  are  to  be  included  in  the  group  mentioned, 
for  many  other  types  of  bacteria,  when  present  in  milk  in  large  numbers, 
may  injure  the  flavor  of  the  butter  prepared  from  it. 

The  acid  fermentation  of  the  cream  is  most  frequently  called  the 
ripening  of  cream  and  sour-cream  butter  is  frequently  called  ripened- 
cream  butter.  The  ripening  of  the  cream  not  only  increases  the  flavor 
of  the  product,  but  it  enhances  its  keeping  quality.  The  ripening  of  the 
cream  also  aids  in  the  mechanical  process  of  churning,  the  sour  cream 
churning  more  easily  and  with  less  loss  of  fat  in  the  butter  milk. 

KINDS  AND  NUMBERS  or  BACTERIA  IN  CREAM. — The  number  and 
kinds  of  bacteria  found  in  cream  are  dependent  upon  the  number  and 
kind  in  the  milk  from  which  the  cream  is  obtained.  The  cream  will, 
however,  contain  a  greater  number  of  bacteria  per  unit  volume  than  the 

*  See  Chap.  I.  Div.  IV,  in  which  the  groups  of  bacteria  are  considered. 


RELATION    OF   MICROORGANISMS    TO  BUTTER  477 

milk,  since  the  immense  number  of  fat  globules  passing  through  the 
milk  serum  carry  mechanically  a  considerable  proportion  of  the  bacteria 
of  the  milk  into  the  cream.  This  phenomenon  is  to  be  noted  in  gravity 
creaming,  but  to  a  much  greater  extent  in  the  removal  of  the  cream  by 
use  of  the  separator. 

SPONTANEOUS  RIPENING  or  CREAM. — By  this  expression  is  meant 
the  fermentation  of  the  cream  by  those  acid-forming  bacteria  that  have, 
from  one  source  and  another,  gained  entrance  to  it,  but  which  have  not 
been  intentionally  added.  Under  these  conditions  the  butter-maker  can 
exert  but  little  control  over  the  fermentation.  A  very  considerable  part 
of  the  butter  made  from  such  cream  has  an  excellent  flavor,  because  at 
the  temperature  at  which  cream  is  usually  kept,  Bact.  lactis  acidi  and 
related  organisms  are  the  primary  factors  concerned  in  its  fermentation 
and  their  by-products  produce  desirable  flavors  in  butter.  It  has  often 
been  asserted  that  the  highest  type  of  butter  can  be  made  only  from 
spontaneously  ripened  cream. 

As  the  cream  from  many  farms  was  assembled  at  a  creamery  for  the 
manufacture  of  butter,  it  became  evident  that  some  means  of  controlling 
the  type  of  fermentation  in  the  cream  was  needed.  If  the  milk  had  been 
produced  under  clean  conditions,  and  had  been  received  at  the  creamery 
before  the  acid  fermentation  had  gone  on  to  any  extent,  and  if  the 
cream  was  then  kept  at  temperatures  most  favorable  for  the  lactic  bac- 
teria, the  product  was  likely  to  be  of  good  quality,  but  such  ideal  condi- 
tions did  not  always  obtain.  Cream  containing  a  large  proportion  of 
harmful  bacteria,  or  in  an  advanced  state  of  fermentation,  or  possessing 
an  undesirable  flavor  was  often  received,  and  the  butter-maker  could 
not  control  the  quality  of  the  product  under  such  conditions. 

USE  OF  CULTURES  IN  BUTTER  MAKING. — As  the  science  of  micro- 
biology progressed  and  the  role  of  microorganisms  in  all  kinds  of  fermen- 
tation became  known,  it  became  evident  that  the  control  of  the  causal 
organism  is  an  important  factor  in  determining  the  quality  of  any  prod- 
uct of  the  fermentation  industries.  In  the  manufacture  of  butter,  the 
first  step  in  this  direction  was  the  addition  of  some  fermented  milk, 
cream,  or  of  buttermilk  to  the  cream  to  be  ripened.  In  this  manner  the 
number  of  acid-forming  organisms  in  the  cream  was  greatly  increased, 
and  the  fermentation  went  on  more  rapidly  and  in  a  more  definite  direc- 
tion than  without  such  additions,  as  the  bacteria  added  were  largely  of 
the  desirable  group.  Bad.  lactis  acidi.  The  addition  of  fermented  milk 


MICROBIOLOGY   OF   MILK  AND   MILK   PRODUCTS 

to  accelerate  the  souring  of  cream  antedates  by  many  hundred  years  the 
science  of  bacteriology. 

The  next  logical  step  in  the  development  of  the  process  was  the  use  of 
the  same  types  of  bacteria  from  day  to  day.  Cultures  of  these  were  ob- 
tained by  allowing  a  quantity  of  milk  to  sour,  and  if  it  had  the  desired 
flavor,  a  small  amount  of  it  was  added  to  another  quantity  of  milk  that 
had  been  heated,  in  order  to  destroy  the  acid-forming  bacteria  it  con- 
tained. By  the  daily  preparation  of  some  heated  milk,  and  the  inocula- 
tion of  it  with  the  soured  milk  previously  prepared,  the  butter-maker 
could  use  the  same  types  of  bacteria  for  an  indefinite  time  for  addition 
to  the  cream. 

It  had  been  found  by  Hansen  that,  in  order  to  control  the  flavor  of 
beer,  pure  cultures  of  yeasts  must  be  used  for  the  fermentation  of  the 
wort.  The  success  of  this  method  in  the  brewing  industry  led  to  the 
introduction  of  pure  lactic  cultures  for  the  fermentation  of  cream.  The 
use  of  such  cultures  was  suggested  independently  by  Storch,  a  Danish 
bacteriologist  and  by  Weigmann,  the  director  of  the  dairy  experiment 
station  at  Kiel  in  Germany,  in  1890.  Many  cultures  were  isolated  and 
tested  as  to  their  effect  on  the  flavor  of  butter.  Those  found  to  be 
desirable  could  be  maintained  in  the  laboratory,  and  could  be  furnished 
to  butter-makers  to  be  used  and  propagated  in  a  manner  similar  to  the 
method  employed  with  the  impure  and  less  constant  home-made  starters. 
The  pure  cultures  of  lactic  bacteria  are  widely  used  at  present  in  the 
butter-producing  countries  of  the  world  and  their  use  is  being  constantly 
extended,  as  butter  makers  come  to  recognize  the  importance  of  con- 
trolling the  ripening  of  cream. 

It  was  found  that  the  butter  made  from  cream  ripened  by  pure  lactic 
cultures  did  not  possess  as  high  a  flavor  as  did  the  finest  butter  made 
from  naturally  ripened  cream.  This  led  to  the  search  for  organisms 
that  could  be  used  alone,  or  together,  with  the  lactic  bacteria,  and  which 
should  give  the  high  flavor  desired.  Such  cultures  were  found,  but 
their  use  did  not  prove  practical,  either  because  they  did  not  maintain 
their  properties  on  continued  cultivation,  or  because  of  their  effect  on 
the  keeping  quality  of  the  product.  The  difference  in  flavor  in  the  case 
of  butter  made  from  naturally  ripened  cream  and  that  from  cream  rip- 
ened by  pure  lactic  cultures  is  undoubtedly  due  to  the  products  of  the 
B.  coli-aero genes  group. 

The  acid  in  spontaneously  soured  milk  is  very  evident  to  the  taste 


RELATION    OF   MICROORGANISMS    TO  BUTTER  479 

when  the  acidity  is  0.6  per  cent  and  above;  the  volatile  acids  formed  by 
the  members  of  the  colon-aerogenes  and  coccus  groups  impart  a  sharp, 
pungent  taste.  In  milk  of  like  acidity  fermented  by  pure  cultures  of 
Bact.  lactis  acidi,  the  acid  is  scarcely  evident  to  the  taste  and  there  is  no 
sharpness,  due  to-  the  absence  of  volatile  acids.  This  same  difference 
appears  in  the  butter  made  from  the  two  kinds  of  milk. 

The  low  flavor  of  the  butter  made  from  cream  ripened  by  pure 
cultures  was  one  of  the  factors  that  prevented  the  rapid  introduction 
of  the  cultures  in  this  country.  The  demands  of  the  butter  market 
have  changed  and  the  mild  flavored  butter,  which  is  now  considered 
to  be  the  finest,  can  be  made  by  the  use  of  pure  cultures  in  the  fermenta- 
tion of  pure  sweet  cream. 

COMMERCIAL  CULTURES. — In  this  country  the  preparation  and 
distribution  of  cultures  for  the  ripening  of  cream  is  largely  in  the 
hands  of  commercial  firms;  hence,  the  term  "commercial  culture" 
is  applied  to  them.  The  different  pure  cultures  are  propagated  in 
the  laboratory  of  the  maker;  they  are  sent  out  either  as  liquid  cultures, 
a  small  mass  of  milk  or  bouillon  inoculated  with  the  organism,  or  in 
a  dry  form,  the  latter  being  prepared  by  mixing  a  culture  of  the  organism 
with  an  inert  substance,  such  as  milk  sugar,  milk  powder,  or  starch, 
and  drying  at  a  low  temperature.  In  a  liquid  the  organisms  are 
exposed  to  the  effects  of  their  own  by-products,  and  the  vitality  of 
the  culture  is  rapidly  lost.  Such  cultures  must  be  used  when  fresh 
in  order  to  give  good  results,  and  they  cannot  be  kept  in  stock  by  the 
manufacturer  or  dealer.  The  resistance  of  Bact.  lactis  acidi  to  desic- 
cation is  great;  it  thus  lends  itself  to  the  preparation  of  the  dry  cultures, 
in  which  the  organisms  remain  in  a  dormant  condition  and  retain 
their  vitality  for  long  periods. 

Most  of  the  cultures  now  sold  are  pure,  as  this  term  is  used  in  bac- 
teriology, still  others  contain  non-acid-forming  organisns  intentionally 
added  or  introduced  accidentally  during  the  process  of  preparation. 
If  the  lactic  bacteria  are  present  in  such  cultures  in  large  numbers, 
the  impurities  are  usually  of  small  practical  significance.  In  the  past 
so-called  "duplex"  cultures  have  been  sold  which  were  supposed  to 
contain  an  acid-forming  organism  and  a  second  organism  that  was 
to  enhance  the  flavor  of  the  product.  Such  cultures  are  no  longer  sold. 

For  the  propagation  in  the  creamery  the  contents  of  the  container 
purchased  are  added  to  a  small  mass  of  milk  that  has  been  heated 


480  MICROBIOLOGY   OF   MILK  AND   MILK   PRODUCTS 

to  destroy  all  non-spore-forming  bacteria  and  other  microorganisms; 
the  milk,  after  being  inoculated,  is  incubated  at  favorable  tempera- 
tures and  when  curdled  can  be  used  for  the  inoculation  of  the  second 
and  larger  quantity.  The  process  of  inoculating  a  quantity  of  milk 
is  carried  out  daily.  It  is  impossible  for  the  butter-maker  to  propagate 
the  culture  in  such  a  way  as  to  maintain  its  original  purity,  but  if  the 
milk  is  heated  sufficiently,  if  all  utensils  are  sterilized,  and  if  the  culture 
is  kept  at  a  temperature  that  is  especially  favorable  for  the  organism, 
the  contamination  that  may  occur  will  not  injure  the  culture  for  prac- 
tical work.  The  cultures  propagated  under  such  conditions  gradually 
deteriorate  and  recourse  must  be  had  sooner  or  later  to  a  fresh  culture. 
The  contamination  that  is  of  the  greatest  significance  is  undoubtedly 
that  with  other  acid-forming  bacteria  rather  than  with  the  forms  that 
remain  in  the  milk  after  heating. 

Many  of  the  cultures  gradually  lose  their  fermentative  properties, 
and  do  not  form  acid  rapidly  and  in  sufficient  amounts  to  insure 
exhaustive  churning  and  to  produce  the  desired  degree  of  flavor  in 
the  product.  Cultures  frequently  become  slimy  or  ropy  on  propaga- 
tion. This  is  not  necessarily  due  to  contamination  with  specific 
slime-forming  organisms  but  rather  to  a  change  in  the  lactic  organism 
itself.  Such  an  abnormality  usually  persists  for  only  a  short  period 
and  the  conditions  that  govern  its  appearance  and  disappearance 
are  not  known.  It  is  asserted  by  practical  butter  makers  that  the 
development  of  too  high  an  acidity  in  the  cultures  as  they  are  propa- 
gated in  the  creameries  permanently  impairs  the  value  of  the 
culture. 

The  cultures  are  propagated  in  skim-milk.  Where  this  is  not  avail- 
able, unsweetened,  condensed  milk  or  milk  powder  have  been  employed. 
Efforts  have  been  made  to  grow  the  bacteria  in  some  other  kind  of 
medium  than  milk,  but  without  success.  The  starter  is  said  to  be 
ripe  or  in  the  best  condition  for  use  soon  after  curdling,  or  when  the 
acidity  is  0.5  to  0.7  per  cent,  as  at  this  time  it  contains  the  maximum 
number  of  living  cells.  The  practical  man  thus  uses  the  curdling 
as  an  indication  of  the  ripeness  of  the  starter.  The  curdled  milk  should 
show  no  free  whey,  and  the  curd  should  be  easily  broken  up  to  form 
a  creamy  mass  that  can  be  uniformly  incorporated  with  the  cream. 
The  temperature  of  incubation  and  the  amount  of  initial  inoculation 
determine  the  rapidity  with  which  the  acid  fermentation  will  progress, 


RELATION    OF    MICROORGANISMS    TO  BUTTER  481 

the  maker  seeking  to  regulate  these  so  that  the  culture  shall  be  ripe  at 
the  desired  time  each  day. 

USE  OF  PURE  CULTURES  IN  RAW  CREAM. — The  cream  as  it  reaches 
the  creamery  contains  a  greater  or  less  number  of  acid-forming  bac- 
teria that  ultimately  will  cause  it  to  ripen  and  the  flavor  of  the  butter 
will  be  due  to  the  by-products  of  the  mixture  of  bacteria.  If,  through 
the  addition  of  a  pure  culture,  the  relative  number  of  organisms  that 
are  known  to  be  favorable  is  greatly  increased,  the  flavor  of  the  product 
should  be  improved.  This  has  been  found  to  be  true  in  practice  and 
it  is  now  believed  that  pure  cultures  are  of  value  not  only  in  the  ripen- 
ing of  sweet  cream,  but  that  the  addition  of  a  relatively  large  amount 
of  starter  to  cream  that  is  already  fermented  will  enhance  the  value 
of  the  butter. 

USE  OF  PURE  CULTURES  IN  PASTEURIZED  CREAM. — It  is  evident 
that  the  maker  has  but  imperfect  control  over  the  fermentative  proc- 
esses when  raw  cream  is  treated  with  a  pure  culture.  To  insure  more 
perfect  control  the  destruction  of  the  contained  bacteria  and  the 
subsequent  inoculation  of  the  cream  with  a  pure  culture  is  indicated. 
The  introduction  of  the  process  of  pasteurization  of  cream  for  butter 
making  was  due  to  Storch.  In  Denmark  this  method  is  used  almost 
exclusively.  It  has  been  introduced  into  the  other  dairy  countries 
of  the  world  and  is  constantly  spreading.  Pasteurization  combined 
with  the  use  of  the  pure  culture  represents  the  highest  type  of  modern 
butter  making,  and  where  the  raw  product  can  be  obtained  in  a  fresh 
condition  the  butter-maker  has  perfect  control  over  the  bacteria 
that  cause  the  ripening;  hence  he  can  control  the  flavor  of  the  butter, 
both  qualitatively  and  quantitatively. 

The  intensity  of  flavor  of  butter  is  dependent  upon  the  amount 
of  acid  that  is  developed  in  the  cream  or  more  correctly  on  the  ratio 
between  the  amount  of  fat  and  the  by-products  of  the  acid  fermenta- 
tion. If  these  by-products  are  small  in  amount,  as  in  cream  having  a 
low  acidity,  the  flavor  of  the  butter  will  be  low.  If  the  acidity  is 
allowed  to  reach  the  maximum,  the  flavor  will  be  much  higher.  Thus 
the  maker  can  control  the  intensity  of  flavor  of  butter  as  accurately 
as  he  can  the  kind  of  flavor.  With  rich  cream,  the  acidity  that  can 
be  developed  is  small  and  the  ratio  between  the  fat  and  the  products 
of  fermentation  is  low;  thus,  the  flavor  of  butter  made  from  very  heavy 
cream  is  certain  to  be  low. 

31 


482  MICROBIOLOGY  OF  MILK  AND  MILK  PRODUCTS 

PURE  CULTURES  IN  OLEOMARGARINE  AND  RENOVATED  BUTTER. — 
It  was  previously  mentioned  that  the  manufacturer  of  butter  substi- 
tutes employs  the  same  methods  to  impart  butter  flavor  to  his  prod- 
ucts as  does  the  butter-maker.  The  oleomargarine  manufacturers 
employ  pure  cultures  of  lactic  bacteria  for  the  fermenting  of  milk 
that  is  mixed  with  the  fats  they  employ.  The  same  practice  is  followed 
by  the  manufacturer  of  renovated  butter.  Many  of  the  creameries 
of  the  western  states  receive  cream  that  is  shipped  long  distances, 
and  is  collected  from  the  farms  but  once  or  twice  a  week.  It  is  thus 
in  an  advanced  state  of  fermentation  when  it  reaches  the  creamery. 
In  order  to  prepare  from  this  grade  of  cream,  which  often  has  a  most 
undesirable  flavor,  a  merchantable  product,  various  means  are  em- 
ployed to  remove  the  flavoring  substances  and  to  replace  them  with 
desirable  flavors  from  the  pure  cultures.  The  acidity  may  be  reduced 
by  the  addition  of  lime  so  that  the  cream  can  be  pasteurized;  the 
cream  may  be  aerated  by  passing  air  through  it,  or  it  may  be  mixed 
with  water  and  reseparated.  After  such  treatment  it  is  mixed  with 
a  large  proportion  of  milk  fermented  by  a  pure  culture  and  churned. 
The  resulting  product  is  constantly  sold  as  the  highest  grade  of  creamery 
butter. 

ABNORMAL  FLAVORS  or  BUTTER. — Most  of  the  abnormal  flavors  of 
butter  are  traceable  to  the  partial  replacement  of  the  desirable  acid- 
forming  bacteria  with  other  types  of  microorganisms  Many  samples 
of  butter  having  abnormal  flavors  have  been  examined,  and  the  organ- 
isms believed  to  be  the  cause  isolated  and  studied  but  it  cannot  be  said 
that  any  particular  group  of  microorganisms  can  be  associated  with  any 
of  the  abnormal  flavors  met.  It  is  asserted  that  "  oily  "  butter,  i.e.,  that 
having  the  taste  of  machine  oil,  is  caused  by  bacteria  and  by  microorgan- 
isms that  decompose  the  fat,  as  Oidium  lactis,  yeasts,  and  liquefying  bac- 
teria. Organisms  of  the  B.  coll  group  that  produce  a  turnip-like  flavor 
in  butter  have  been  described  by  Weigmann.  The  flavors  of  putrid 
butter,  fishy  butter  and  also  many  other  abnormal  flavors  have  been 
ascribed  to  bacteria. 

Other  abnormal  flavors  may  be  due  to  the  presence  in  the  milk  of 
certain  aromatic  principles  contained  in  the  feed  and  excreted  in  the 
milk.  Cabbage,  turnips,  and  other  plants  impart  their  characteristic 
taste  to  the  milk  and  butter. 


RELATION   OF   MICROORGANISMS   TO  BUTTER  483 

DECOMPOSITION  PROCESSES  IN  BUTTER 

Butter  is  a  finished  product  at  the  time  it  is  removed  from  the  mod- 
ern churn  and  all  subsequent  changes  are  likely  to  cause  more  or  less 
deterioration.  The  specific  causes  of  these  changes  are  not  well  known 
but  it  is  very  evident  from  a  study  of  the  conditions  that  favor  or  retard 
the  appearance  of  the  flavors,  characterizing  these  changes,  that  biolog- 
ical factors  are  concerned.  When  raw  cream  is  used,  sweet-cream  butter 
has  very  poor  keeping  qualities.  As  the  proportion  of  acid-forming 
bacteria  in  butter  is  increased,  either  by  the  fermentation  of  the  cream, 
by  the  addition  of  pure  cultures,  and  through  the  use  of  the  latter  in 
connection  with  pasteurization,  the  keeping  qualities  are  enhanced. 
Of  the  butter  made  from  ripened  cream,  that  prepared  from  cream, 
handled  in  a  clean  manner,  and  thoroughly  pasteurized  and  ripened  with 
a  pure  culture  of  Bact.  lactis  acidi,  has  the  best  keeping  quality.  If. 
fresh,  sweet,  clean  cream  is  pasteurized,  the  butter  will  have  better 
keeping  quality  than  when  made  from  the  same  cream  pasteurized  and 
ripened  with  a  pure  culture.  This  is  evidence  that  not  only  the  bac- 
teria other  than  Bact.  lactis  acidi  are  harmful,  but  that  this  organism, 
that  has  usually  been  considered  without  influence  on  the  keeping 
quality,  must  be  classed  as  one  of  the  factors  hi  the  decomposition  of 
butter. 

It  has  been  shown  that  the  bacterial  content  of  the  water  used  for 
the  washing  of  the  butter  has  an  influence  on  the  keeping  quality.  If 
the  water  is  of  surface  origin  and  contains  the  bacteria  peculiar  to  these 
types  of  waters,  its  influence  may  be  marked  and  some  method  of  treat- 
ment must  be  followed.  Filtering  or  heating  the  water  has  been  re- 
sorted to,  the  latter  with  marked  success.  A  pure  water  will  contain 
so  few  bacteria  that  they  will  not  exert  any  noticeable  influence  on  the 
keeping  quality  of  the  butter. 

Storage  temperature  also  has  a  marked  influence  on  the  deterioration 
changes  in  butter.  Modern  butter-storage  rooms  are  kept  below 
o°F.;  the  butter  deteriorates  slowly  during  storage  at  these  tempera- 
tures, but  on  removal  undergoes  change  much  more  rapidly  than 
would  have  been  true  before  storage.  Another  factor  that  is  of  influence 
in  the  keeping  of  butter  is  the  amount  of  salt  used.  In  salted  butter,  the 
contained  water  is  a  concentrated  brine;  in  such  a  medium  most  forms 
of  bacteria  are  unable  to  grow.  Small  packages  deteriorate  more 


484  MICROBIOLOGY   OF   MILK  AND   MILK  PRODUCTS 

rapidly  than  large  ones,  because  the  proportion  of  the  mass  of  butter 
exposed  to  the  air  is  relatively  greater.  Exposure  to  light  is  also 
claimed  to  exert  a  harmful  influence.  Antiseptic  substances  such  as 
borax  and  boric  acid  have  a  marked  effect  on  the  deterioration  changes. 
The  New  Zealand  and  Australian  butter  exported  to  the  English  mar- 
kets is  treated  with  preservatives. 

A  large  amount  of  experimental  work  has  been  done  in  order  to 
determine  the  effect  of  specific  organisms  on  the  keeping  quality  of 
butter.  The  results  obtained  have  not  been  definite  and  it  is  not  certain 
that  the  organisms  employed  are  constantly  concerned  in  the  deteriora- 
tion changes.  It  is  very  probable  that  both  bacteria  and  molds  exert 
an  influence.  The  chemical  changes  that  take  place  in  the  spoiling  of 
butter  are  no  better  known  than  are  the  causal  factors.  It  has  been 
asserted  that  there  is  a  decomposition  of  the  glycerides  with  a  resulting 
increase  in  free  acids.  It  has  been  shown  that  this  does  not  always 
occur;  that  a  butter  may  be  in  an  advanced  state  of  decomposition  and 
its  content  in  volatile  acids  not  be  higher  than  when  fresh.  Two  types 
of  changes  are  usually  distinguished,  rancidity  and  the  appearance  of  a 
tallow-like  odor.  The  latter  may  be  due  to  purely  chemical  factors, 
while  the  former  is  quite  certainly  biological. 

Moldy  butter  is  a  frequent  trouble  encountered  by  the  butter-maker. 
If  the  butter  is  not  salted,  molds  may  develop  just  below  the  surface. 
The  most  usual  form  of  mold  to  appear  is  one  with  black  hyphae;  the 
slightest  development  of  which  will  be  evident  on  the  butter.  In  the 
case  of  salted  butter,  mold  on  the  butter  itself  is  very  rare,  due  appar- 
ently to  the  concentration  of  the  brine  in  the  butter.  The  parchment 
paper  in  which  print  butter  is  wrapped  and  with  which  the  butter  con- 
tainers are  lined  is  an  excellent  substratum  for  mold  growth.  If  the 
papers  and  containers  are  badly  contaminated  with  mold  spores,  or 
if  they  have  been  kept  under  such  conditions  as  to  permit  of  a  limited 
amount  of  growth  before  they  are  used,  the  development  of  the  mold  on 
the  paper  after  it  is  brought  into  contact  with  the  butter  is  likely  to  be 
rapid,  even  at  low  temperatures,  and  the  butter, is  likely  to  reach  the 
market  in  an  objectionable  condition.  The  paper  may  be  rendered 
free  from  molds  by  placing  it  in  water  which  has  been  heated  to  at  least 
80°.  Butter  tubs  are  scalded,  steamed,  or  soaked  in  brine  or  treated 
with  a  dilute  solution  of  formalin  in  order  to  destroy  the  mold  spores 
that  may  be  present.  The  most  efficient  manner  of  preventing  trouble 


RELATION    OF    MICROORGANISMS    TQ  BUTTER  485 

is  to  coat  the  inside  of  the  butter  tub  with  paraffin.  This  prevents 
trouble  from  the  container  but  not  from  the  paper.  Mold  spores  or 
vegetative  hyphse  are  likely  to  be  present  in  the  cream.  Those  which 
find  themselves  on  the  immediate  surface  of  the  butter  may  grow.  This 
source  of  trouble  can  be  overcome  by  pasteurization  of  the  cream. 

PATHOGENIC  BACTERIA  IN  BUTTER 

If  the  milk  contains  pathogenic  bacteria,  they  are  certain  to  pass 
into  the  cream  and  be  incorporated  in  the  butter.  It  is  not  believed 
that  butter  is  an  important  agent  in  the  distribution  of  the  organisms 
of  tuberculosis  and  typhoid  fever,  although  both  are  able  to  exist  in 
salted  butter  for  over  two  months.  Foot-and-mouth  disease  is  said 
be  caused  in  humans  by  the  use  of  butter  made  from  the  milk  of 
infected  animals,  but  this  may  still  be  regarded  as  a  mooted 
question. 


CHAPTER  III* 

THE  RELATION  OF  MICROORGANISMS  TO  CHEESE 

GENERAL 

Cheese  consists  of  the  fat  and  casein  of  milk,  together  with  the 
insoluble  salts;  however,  along  with  these  constituents  are  carried 
some  of  the  moisture  of  milk,  in  which  are  dissolved  small  quantities 
of  sugar,  albumin,  and  salts.  The  amount  of  moisture  and  soluble 
constituents  found  in  cheese  is  determined  by  the  amount  of  whey 
incorporated  in  the  curd. 

In  the  process  of  making  cheese,  it  is  necessary  to  curdle  the  milk, 
thus  enabling  the  separation  of  the  casein  and  fat  from  the  milk  serum. 
Two  methods  are  employed  to  accomplish  this  purpose,  and,  as  a 
result,  two  types  of  cheeses  are  produced. 

TYPES  OF  CHEESE 

These  types  may  be  designated  as  "Acid-curd  Cheeses"  and  "Rennet- 
curd  Cheeses" 

ACID-CURD  CHEESES. — The  curdling  may  be  accomplished  by 
allowing  the  milk  to  undergo  acid  fermentation,  either  spontaneously 
through  the  action  of  the  normal  flora  of  the  milk,  or  through  the 
addition  of  pure  lactic  cultures.  Most  acid-curd  cheeses  are  ready 
for  use  as  soon  as  the  whey  has  been  removed  by  draining  and  the 
curds  salted.  Acid-curd  cheeses  are  not  commercially  important. 
They  are  made  for  local  consumption  and  are  to  be  classed  as  a  form 
of  sour  milk.  They  owe  their  flavor  to  the  products  of  the  acid  fer- 
mentation, especially  lactic  acid.  The  moisture  content  is  high, 
which,  together  with  the  acid  reaction,  favors  the  growth  of  molds 
and  yeasts.  These  biological  agents  may  soon  spoil  the  cheese. 

RENNET-CURD  CHEESES. — All  of  the  important  varieties  of  cheeses 
are  made  by  the  use  of  rennet  for  the  curdling  of  the  milk.  Over 

*  Prepared  by  E.  G.  Hastings. 

486 


RELATION    OF    MICROORGANISMS    TO    CHEESE  487 

four  hundred  kinds  of  rennet-curd  cheeses  are  made,  but  only  twelve 
to  fifteen  are  of  great  commercial  importance.  With  few  exceptions, 
they  are  made  from  cow's  milk.  From  the  same  raw  material — milk, 
rennet,  and  salt — therefore,  a  wide  variety  of  products,  differing 
in  texture,  taste  and  odor,  is  obtained.  This  fact  indicates  the  im- 
portance of  biological  factors  in  the  changes  the  curd  undergoes  during 
the  ripening  process. 

The  rennet-curd  cheeses  may  be  divided  into:  (i)  hard  cheeses; 
(2)  soft  cheeses;  the  initial  difference  is  largely  in  the  amount  of  whey 
left  in  the  curd  during  the  making  of  the  cheese.  The  two  great  groups 
of  rennet-curd  cheeses  gradually  merge  into  each  other  in  varieties 
that  by  some  are  classed  as  hard  cheese,  by  others  as  soft  cheese. 

The  rennet-cur  d  cheeses,  as  a  rule,  are  at  first  tough  and  rubber-like 
in  .  texture.  The  curd,  which  is  not  easily  digested,  is  quite  insoluble 
in  water  and  is  devoid  of  flavor  and  aroma.  The  curd  must  pass 
through  a  complete  series  of  chemical  and  physical  changes,  which 
alter  its  texture,  solubility,  and  digestibility,  and  give  to  it  a  flavor 
and  aroma  by  which  the  different  kinds  of  rennet-curd  cheeses  are 
especially  to  be  differentiated. 

In  the  hard  cheeses  the  factors  concerned  in  these  changes  act  in  a 
uniform  manner  throughout  the  entire  mass  of  the  cheese,  making 
it  possible  to  manufacture  such  cheeses  in  any  desired  size.  In  the 
case  of  the  soft  cheeses,  the  ripening  changes  are  largely  due  to  agents 
which  grow  only  on  the  surface;  the  products  of  such  agents  by  means 
of  diffusion  gradually  affect  the  entire  mass.  In  order  that  this  may 
take  place  within  a  reasonable  time,  it  is  essential  that  these  cheeses 

made  in  small  sizes.  Then,  too,  the  soft  texture  of  such  cheeses 
makes  it  impossible  to  handle  them  commercially  in  large  sizes. 

CONDITIONS  AFFECTING  THE  MAKING  OF  CHEESE 

QUALITY  OF  MILK. — In  the  curdling  of  milk  by  rennet  the  solid 
Bodies  present  in  the  milk  are  retained  in  the  curd,  thus  the  fat 
globules  are  held,  as  are  also  the  bacteria.  The  latter  continue  to 
grow  as  they  would  have  done  in  the  milk  except  that  growth 
must  take  place  in  the  form  of  colonies  as  in  the  solid  culture  media 
of  the  bacteriologist.  The  bacteria,  however,  produce  the  same  fer- 
mentation in  the  curd  as  they  would  have  done  in  the  uncurdled  milk. 


488  MICROBIOLOGY   OF   MILK   AND    MILK   PRODUCTS 

The  butter-maker  can  control,  through  pasteurization  and  the  use 
of  pure  lactic  cultures,  the  fermentation  of  the  cream.  The  pasteuri- 
zation may  be  so  efficient  as  to  destroy  all  non-spore-forming  bacteria 
since  the  quality  of  the  product  will  not  be  impaired  by  the  use  of 
temperatures  approximating  the  boiling  point.  The  cheese-maker 


i 

r"  :»• 


FIG.  146. — The  type  of  curd  obtained  from  milk  in  which  the  acid-forming  flora 
consi  ts  largely  of  organisms  of  the  B.  coli-aero genes  group.  Many  gas  holes  and 
few  irregular  shaped,  angular,  mechanical  holes  due  to  imperfect  "matting." 
(Original.) 

is  much  more  dependent  on  the  original  quality  of  the  milk,  since  it 
has  not  been  found  possible  to  make  most  of  the  important  varieties  of 
cheeses  from  pasteurized  milk.  If  undesirable  forms  of  microorganisms 
are  present  in  the  milk,  they  will  pass  into  the  cheese  and  there  produce 
their  harmful  effects.  Through  the  addition  of  pure  cultures  of 


RELATION  OF  MICROORGANISMS  TO  CHEESE       489 

Bact.  lactis  acidi  to  the  milk,  the  proportion  of  desirable  bacteria  can 

be  increased  and  a  partial  control  of  the  fermentation  thus  secured. 

TESTS  FOR  THE  QUALITY  OF  MILK. — Methods  by  which  the  cheese 

maker  can  determine,  in  a  rough  manner,  the  kinds  of  bacteria  present 


FIG.  147. — The  type  of  curd  obtained  from  milk  in  which  the  acid-forming  flora 
consists  almost  wholly  of  Bact.  lactis  acidi.  No  gas  holes  and  no  marked  mechanical 
holes  as  the  curd  has  "matted"  almost  perfectly.  (Original.) 


have  been  devised.     The  bacteria  most  dreaded  and  most  frequently 
present  are  those  of  the  B.  coli-aero genes  group. 

The  method  most  frequently  used  for  their  detection  consists  in 
incubating  a  sample  of  the  milk  to  be  tested  at  temperatures  ranging 
from  35°  to  40°  for  a  few  hours  and  noting  the  type  of  curd  that  is 
formed.  Milk  suitable  for  cheese  making  should  show  the  solid  curd 
characteristic  of  the  Bact.  lactis  acidi  group,  while  gassy  curds  or  soft 


MICROBIOLOGY   OF  MILK  AND   MILK  PRODUCTS 

and  partially  digested  curds  are  indicative  of  bacteria  that  are  likely  to 
be  harmful  in  the  cheese. 

An  improvement  over  the  fermentation  test  of  foreign  origin  has 
been  devised  by  Babcock  and  Russell  and  is  known  as  the  Wisconsin 
Curd  Test.  It  has  for  its  basis  the  same  principle  as  the  simple  fermen- 
tation test;  however,  a  modification  is  introduced;  the  milk  is  curdled  by 
the  addition  of  rennet  and  the  curd  is  cut  and  drained  to  free  it  from  the 
whey  as  completely  as  possible. 

The  undesirable  organisms  most  likely  to  be  present  in  milk  are 
those  of  the  B.  coli-aerogenes  group;  therefore  the  jars  containing  the 
curds  should  be  kept  at  temperatures,  35°  to  40°,  that  will  favor  their 
development.  The  great  advantage  of  the  Wisconsin  Curd  Test  is  its 
greater  delicacy,  since  the  bacteria  are  concentrated  in  a  small  volume, 
and  thus  their  presence  is  more  evident  than  would  be  the  case  in  the 
larger  mass  of  curd  obtained  when  no  rennet  is  added.  The  curd  can 
also  be  removed  from  the  jar,  cut,  tasted,  and  its  texture  determined,  all 
of  which  aid  in  judging  the  quality  of  the  milk.  The  curd  should  have 
a  clean  acid  odor  and  taste;  it  should  be  free  from  sliminess  on  the  sur- 
face, and  possess  a  uniform  texture.  Such  a  curd  can  be  obtained  only 
in  the  presence  of  a  considerable  number  of  lactic  bacteria.  Very 
clean,  fresh  milk  is  likely  to  give  an  undesirable  result,  since  milk 
always  contains  microorganisms  which  will  grow  rapidly  at  the  high 
temperature  in  the  absence  of  the  acid-forming  bacteria  and  which  will 
usually  produce  undesirable  flavors  in  the  curd.  This  fact  should  be 
kept  in  mind  in  the  testing  of  market  milk. 

RIPENING  OF  MILK.*— The  methods  for  the  determination  of  acidity 
in  milk  have  very  considerable  limits  of  error.  It  is  not  possible  to 
detect  any  increase  in  acidity  until  the  number  of  acid-forming  bacteria 
has  increased  to  hundreds  of  thousands  per  c.c.  Originally  it  was 
thought  that  no  acid  was  produced  by  the  growth  of  the  acid-forming 
bacteria  during  the  initial  stages  of  their  development.  This  period 
during  which  bacterial  proliferation  was  taking  place,  but  without  an 
apparent  increase  in  acidity,  was  known  as  the  "  period  of  incubation." 
It  is  now  certain  that  this  rests  upon  our  inability  to  detect  small 

*  In  order  to  illustrate  the  r61e  of  microorganisms  in  the  making  and  ripening  of  cheeses,  a 
somewhat  detailed  summary  of  the  present  knowledge  concerning  their  action  in  Cheddar  cheese 
will  be  given.  Many  of  the  factors  concerned  in  the  ripening  of  this  kind  of  cheese  also  function 
in  the  ripening  of  other  rennet  cheeses.  In  their  description  only  such  additional  factors  need 
be  considered  as  are  not  active  in  Cheddar  cheese. 


RELATION   OF   MICROORGANISMS    TO   CHEESE  491 

increases  in  acidity.  The  Cheddar  cheese-maker  desires  milk  that  shall 
contain  such  a  number  of  acid-forming  bacteria  that  during  the  opera- 
tions that  are  carried  on  in  the  first  part  of  the  cheese-making  process 
large  amounts  of  acid  shall  be  formed  in  the  curd.  He  thus  wishes  to 
know,  as  accurately  as  can  be  determined  under  the  conditions  found 
in  the  factory,  the  number  of  bacteria  in  the  milk  which  he  is  to  use. 
This  information  is  gained  either  by  the  titration  of  the  milk  with  a 
standard  alkali  solution  or  by  determining  the  time  required  for  the 
curdling  of  a  definite  quantity  of  milk  at  a  definite  temperature  by  a 
known  quantity  of  rennet.  Very  much  smaller  increases  in  acidity 
can  be  detected  by  the  so-called  rennet  test  than  by  titrating 
the  milk.  If  the  milk  shows  the  desired  acidity  when  it  reaches  the 
factory,  the  making  process  is  immediately  begun.  If  the  milk  is  too 
sweet,  or  in  other  words,  too  low  in  its  bacterial  content,  bacterial 
growth  is  favored  by  warming  the  milk  to  temperatures  most  favorable 
for  the  lactic  bacteria,  30°  to  32°,  and  by  the  addition  of  pure  cultures 
of  Bad.  lactis  acidi  which  are  identical  in  nature  and  the  method  of 
propagation  with  those  used  in  butter  making.  The  development  of  a 
slight  acidity  is  known  as  the  "ripening"  of  milk. 

In  order  to  insure  proper  rennet  action  the  maker  of  Cheddar  cheese 
desires  the  milk  to  have  an  acidity  of  about  0.2  per  cent.  He  thus 
wishes  milk  in  which  an  appreciable  amount  of  acid  has  been  formed. 

CURDLING  OF  MILK. — Under  the  influence  of  a  favorable  tempera- 
ture and  the  slight  acidity,  the  milk  is  quickly  changed  by  the  rennin*  to 
a  firm,  jelly-like  mass  that  is  cut,  with  appropriate  knives,  into  small 
cubes.  The  curd  encloses  over  80  per  cent  of  the  bacteria  in  the  milk. 
The  same  factors  that  favor  the  curdling  of  the  milk  favor  the  shrinking 
of  the  curd  and  the  expulsion  of  the  whey  from  the  cubes.  The  develop- 
ment of  acid  within  the  curd  is  rapid,  due  to  the  concentration  of  large 
numbers  of  bacteria  in  a  small  volume  and  to  the  favorable  environ- 
ment. During  the  six  to  eight  hours  that  elapse  between  the  curdling 
of  the  milk  and  the  pressing  of  the  curd,  the  increase  of  acidity  is  over 
o.i  per  cent  per  hour.  The  following  table  gives  the  acidity  of  milk  and 
the  whey  expressed  from  the  curd  at  various  stages  in  the  making  of  a 
typical  Cheddar  cheese. 

•  The  rennet  used  in  cheese-making  is  obtained  by  extracting  the  abomasum,  the  true  diges- 
tive stomach  of  the  calf,  with -a  solution  of  sodium  chloride.  The  extract  contains  two  enzymes, 
a  clotting  or  curdling  enzyme,  rennin,  and  a  proteolytic  enzyme,  Pepsin. 


4Q2  MICROBIOLOGY   OF   MILK  AND   MILK  PRODUCTS 

Acidity  of  milk  before  adding  rennet o. 20-0. 21  per  cent. 

Acidity  of  whey  immediately  after  cutting  curd.  o.  14-0. 145  per  cent. 
Acidity  of  whey  when  removed  from  the  curd. .  o.  16-0. 18  per  cent. 

Acidity  of  whey  when  curd  is  packed o .  24-0 . 30  per  cent. 

Acidity  of  whey  when  curd  is  milled 0.65-0. 75  per  cent. 

Acidity  of  whey  when  curd  is  salted 0.90-1 . 10  per  cent. 

MANIPULATION  OF  THE  CURD.* — The  curd  particles  at  first  show 
little  tendency  to  cohere;  but,  as  the  acidity  increases,  the  nature  of  the 
curd  changes,  and,  when  the  whey  is  removed,  the  pieces  of  curd  soon 
cohere  and  ultimately  form  a  single  mass  in  which  the  original  cubes  of 
curd  cannot  be  detected.  The  fusion  of  the  curd  particles  is  known  as 
" matting"  and  is  an  important  step  in  the  Cheddar  process.  The  lack 
of  acid  formation  within  the  curd  prevents  matting  while  the  curd  is  in 
the  vat,  and  may  even  render  difficult  the  fusion  of  the  particles  under 
pressure.  The  nature  of  the  change  which  the  curd  undergoes  at  this 
stage  in  the  manufacture  is  not  well  understood,  but  probably  is  due  to 
a  combination  between  the  paracasein  and  the  lactic  acid,  the  resulting 
compounds  differing  from  the  paracasein  in  physical  properties  and  in 
solubilities. 

RIPENING  OF  CHEESE. — Cheese  in  ripening  undergoes  profound 
physical  and  chemical  changes  under  the  influence  of  a  number  of 
factors,  which  for  purposes  of  discussion  may  be  divided  into  two  groups : 
those  by  which  the  content  of  soluble  nitrogen  in  the  cheese  is  increased 
and  the  digestibility  enhanced;  and  those  which  cause  the  formation  of 
flavoring  substances.  During  the  ripening  of  the  cheese  the  maker 
can  do  little  toward  the  control  of  the  factors  which  ultimately  deter- 
mine its  commercial  value.  As  in  butter,  the  flavor  is  the  most  impor- 
tant characteristic  of  the  ripened  cheese  and  the  most  difficult  to 
control. 

Theories  of  Cheese  Ripening. — Many  theories  have  been  advanced 
to  explain  the  changes  that  occur  during  the  ripening  process.  Duclaux, 
a  French  microbiologist,  studied  the  bacterial  flora  of  Cantal  cheese 
by  aid  of  the  crude  methods  available  before  the  introduction  of  the 
gelatin-plate  method.  By  the  use  of  the  dilution  method,  using 
bouillon  as  the  nutrient  medium,  he  isolated  a  number  of  kinds  of 
spore-forming  bacteria.  The  organisms  formed  two  enzymes,  one 
a  curdling  enzyme  related  to  rennin,  the  other  a  proteolytic  enzyme 

*  Cheddar  cheese. 


RELATION    OF   MICROORGANISMS   TO   CHEESE  493 

to  which  was  given  the  name  casease.  A  chemical  study  of  the  by- 
products of  the  organisms,  when  growing  in  milk,  revealed  a  number 
of  compounds  that  had  previously  been  found  in  ripe  cheese,  such  as 
leucin,  tyrosin,  and  the  ammonia  salts  of  acetic,  valeric  and  carbonic 
acids.  The  cultures  often  possessed  a  cheese-like  odor.  These  facts 
led  Duclaux  to  believe  this  class  of  organisms  was  responsible  for  the 
ripening  of  the  hard  cheese  in  question.  The  generic  name  Tyrothrix 
was  applied  on  account  of  the  supposed  relation  to  cheese.  This  term  is 
still  found  in  current  bacteriological  literature.  The  methods  employed 
by  Duclaux  were  such  as  favored  the  growth  of  the  liquefying,  rather 
than  the  acid-forming  bacteria.  To  the  latter  more  recent  investi- 
gators have  devoted  attention. 

The  theory  that  the  proteolytic  bacteria  function  in  the  ripening 
of  hard  cheese  has  been  more  recently  emphasized  by  Adametz.  It 
is  sufficient  to  say  that  the  number  of  spore-forming  proteolytic  bac- 
teria in  cheese  is  not  sufficiently  large,  nor  is  their  presence  so  constant 
that  any  importance  can  be  attached  to  them.  Any  agent  to  be  con- 
sidered as  a  factor  in  the  ripening  process  must  be  present  in  every 
cheese  in  sufficient  numbers  to  account  for  the  change  for  which  it  is 
considered  responsible.  Such  agents  should  be  capable  of  demonstra- 
tion. It  should  be  remembered  that,  by  following  the  rules  laid  down 
by  the  practical  maker,  a  normal  cheese  can  invariably  be  made, 
hence  the  factors  of  importance  in  the  ripening  must  be  constantly 
present  in  the  milk  or  rennet.  It  is  doubtful  whether  the  liquefying 
bacteria  will  satisfy  this  requirement.  It  has  been  shown  by  de 
Freudenreich  that  such  organisms,  even  when  added  to  milk  in  large 
numbers,  exert  no  influence  on  the  ripening  of  hard  cheese,  since  the 
conditions  within  the  cheese  are  not  such  that  growth  can  occur. 

De  Freudenreich,  a  Swiss  microbiologist,  by  the  aid  of  modern 
methods,  demonstrated  the  constant  presence  of  certain  classes  of 
acid-forming  bacteria  in  Swiss  cheese,  and  to  them  ascribed  an  impor- 
tant role  in  the  ripening  of  this  hard  cheese.  He  was  led  to  this  con- 
clusion by  their  great  numbers  in  the  fresh  cheese,  and  by  the  fact 
that  cheese  made  from  milk  drawn  under  aseptic  conditions,  which 
thus  contains  no  lactic  bacteria,  do  not  ripen;  through  the  discovery, 
also,  that  certain  of  the  lactic  bacteria,  predominating  in  Swiss  cheese, 
those  of  the  Bad.  bulgaricum  group,  exert  a  solvent  effect  on  the 
casein  of  milk,  although  they  are  devoid  of  action  on  gelatin. 


494  MICROBIOLOGY   OF   MILK  AND'  MILK  PRODUCTS 

Babcock  and  Russell  demonstrated  the  presence  of  an  inherent 
proteolytic  enzyme  in  milk,  to  which  the  term  galactase  was  applied. 
This  enzyme  can  be  demonstrated  by  preserving  a  sample  of  fresh 
milk  with  chloroform  or  other  mild  antiseptic.  At  37°  curdling 
occurs  in  three  to  four  weeks;  the  content  of  soluble  nitrogen  in  the 
milk  is  slowly  augmented.  The  presence  of  this  proteolytic  enzyme, 
together  with  the  fact  that  a  normal  cheese  cannot  be  made  from  milk 
in  which  this  enzyme  has  been  destroyed  by  heat,  led  these  investi- 
gators to  consider  this  inherent  enzyme  of  milk  an  important  factor 
in  cheese  ripening. 

Present  Knowledge  of  Causal  Factors* — The  role  of  certain  factors 
in  the  ripening  of  Cheddar  cheese  has  been  established  beyond  doubt 
by  the  chemical  and  bacteriological  investigations  of  recent  years. 
It  is  certain  that  acid-forming  bacteria  are  essential  factors  in  the 
ripening  of  this  kind  of  hard  cheese,  and  probably  of  all  kinds  of  rennet 
cheeses. 

As  has  been  shown  the  growth  of  acid-forming  bacteria  is  rapid 
during  the  making  of  Cheddar  cheese.  The  growth  continues  during 
the  pressing  and  subsequent  thereto;  the  maximum  number  of  lactic 
bacteria  is  found  when  the  cheese  is  one  to  five  days  old.  As  many  as 
1,500,000,000  per  g.  of  the  moist  cheese  have  been  demonstrated. 

Causes  of  Proteolysis. — The  proteolytic  action  of  rennet  extract  on 
the  paracasein  of  cheese  was  demonstrated  by  Babcock  and  Russell, 
and  by  Jensen.  This  property  is  due  to  the  fact  that  rennet  extract 
also  contains  the  enzyme  pepsin,  which  for  its  action  outside  the  body 
requires  conditions  similar  to  those  which  obtain  in  the  stomach; 
in  other  words,  the  presence  of  sufficient  acid  to  activate  it.  The  hydro- 
chloric acid  secreted  by  the  walls  of  the  stomach  acts  as  the  activating 
agent  in  the  body.  The  acidity  resulting  from  the  fermentation  of 
the  sugar  in  the  curd  is  sufficient  to  activate  the  pepsin.  Under  its 
influence  the  paracasein  is  partially  converted  into  soluble  decomposi- 
tion products  such  as  albumoses  and  peptones.  In  the  absence  of 
acid-forming  bacteria  no  acid  is  formed;  consequently  the  pepsin  does 
not  become  active  and  no  proteolytic  effect  is  produced.  Under  these 
conditions  the  curd  remains  tough  and  elastic  and  the  solubility  is 
not  increased.  It  is  thus  evident  that  acid-forming  bacteria  are  essen- 
tial factors  in  cheese  ripening.  The  pepsin  of  the  rennet  extract  and 

*Cheddar  cheese. 


RELATION    OF    MICROORGANISMS    TO    CHEESE  495 

the  galactase  suffice  to  account  for  the  initial  proteolysis  of  the  para- 
casein.  Since  neither  of  these  enzymes  forms  ammonia,  which  is 
always  found  in  ripe  cheeses,  some  other  factor  must  be  responsible 
for  the  production  of  this  compound.  It  may  owe  its  origin  to  micro- 
organisms not  yet  discovered. 

Prevention  of  Putrefaction. — The  various  stages  in  the  decomposition 
of  milk  have  been  outlined  in  a  previous  chapter.  Briefly  they  are  as 
follows :  The  first  evident  change  is  the  curdling  due  to  the  acid-forming 
bacteria.  Succeeding  this,  the  acid,  semi-solid  mass  or  curd  is  a  favor- 
able substratum  for  the  characteristic  mold  of  milk,  Oidium  lactis,  which 


A  B 

FIG.  148. — Proteolytic  action  of  rennet  extract  in  the  absence  and  in  the  presence 
of  acid-forming  bacteria.  A,  sterile  milk  agar;  a  strip  of  filter-paper  treated  with 
rennet  was  allowed  to  remain  on  the  medium  for  one  hour  at  37°.  No  digestion  of  the 
casein.  B,  milk  agar  inoculated  with  Bact.  lactis  acidi;  incubated  for  twenty-four 
hours  at  37°,  then  treated  as  A.  True  digestion  of  the  casein  is  indicated  by  the 
clearing.  (Original.) 

soon  forms  a  white,  velvet-like  layer  over  the  surface  of  the  milk.  Like 
other  molds,  this  form  can  use  acids  as  a  source  of  energy.  The  acid  is 
then  oxidized  to  carbon  dioxide  and  water,  and  thus  the  reaction  of  the 
milk  is  slowly  changed  until  a  point  is  reached  which  allows  the  putre- 
factive bacteria,  that  have  remained  dormant  during  the  period  of  un- 
favorable environment,  to  develop.  The  curd  is  accordingly  pepton- 
ized  and  putrefaction  occurs.  If  the  acid  reaction  is  maintained 
through  the  prevention  of  mold  growth,  the  milk  will  be  preserved  from 


496  MICROBIOLOGY   OF   MILK   AND    MILK  PRODUCTS 

the  attacks  of  putrefactive  organisms  and  will  remain  unchanged  for  an 
unlimited  time. 

The  second  r61e  of  the  acid-forming  bacteria  in  cheese  is  to  protect 
it  against  the  putrefactive  organisms  that  are  constantly  present  in 
milk  and  hence  in  cheese.  The  acid  reaction  of  the  cheese,  due  to  the 
persistence  of  lactic  acid,  or  to  the  formation  of  volatile  acids  after  the 
initial  fermentation,  is  sufficient  to  prevent  the  growth  of  the  putrefac- 
tive bacteria  within  the  cheese.  If  the  cheese  is  made  from  milk  which 
contains  no  acid-forming  bacteria  and  few  putrefactive  ones,  or  if  the 
sugar  is  removed  from  the  curd  by  washing  it  with  water,  the  cheese  will 
not  ripen  since  there  is  no  acid  to  activate  the  pepsin;  the  curd  will  re- 
main in  much  the  same  condition  as  when  it  was  removed  from  the 
press.  Cheese  made  from  milk  containing  no  acid-forming  bacteria  but 
many  putrefactive  bacteria  is  likely  to  undergo  putrefaction,  since  the 
latter  class  of  organisms  finds  conditions  for  growth  in  the  absence  of 
an  acid  reaction.  Such  a  condition  is  rarely  noted  in  a  hard  cheese 
under  normal  conditions,  but  may  be  produced  experimentally.  The 
biological  acid  may  be  replaced  by  other  acids  added  to  the  curd  in 
appropriate  amounts,  since  these  will  activate  the  pepsin  and  protect 
the  cheese  against  the  attacks  of  putrefactive  bacteria;  but  it  is  not  cer- 
tain that  the  cheese  will  develop  a  normal  flavor  when  lactic  acid  is 
replaced  by  mineral  acids. 

Other  Groups  of  Bacteria  in  Cheese. — It  has  been  shown  at  the  Wis- 
consin Experiment  Station  that  other  groups  of  bacteria  are  constantly 
present  in  Cheddar  cheese.  The  development  of  certain  members  of  the 
Bact.  bulgaricum  group  occurs  somewhat  later  than  that  of  the  Bad. 
lactis  acidi  group.  It  occurs  largely  after  the  sugar  has  disappeared. 
Their  numbers  approximate  those  of  the  Bact.  lactis  acidi  group. 
Coccus  forms  are  also  found  in  great  numbers  in  the  cheese.  It  is 
probable  that  these  two  groups  may  be  responsible  for  the  ammonia 
production,  since  typical  cultures  of  both  groups  are  able  to  produce 
small  amounts  of  ammonia  in  sterile  milk. 

Flavor  Production  in  Cheese. — The  factors  that  have  been  discussed 
are  undoubtedly  the  most  important  ones  concerned  in  the  proteolysis 
of  the  curd,  and  are  thus  the  factors  concerned  in  the  changes  of  texture, 
solubility  and  digestibility.  The  flavor,  which  develops  during  the 
ripening  process,  has  been  regarded  as  due  to  the  proteolysis  of  the  para- 
casein.  A  thoroughly  ripened  cheese  contains  a  large  amount  of  am- 


RELATION   OF   MICROORGANISMS    TO   CHEESE 


497 


monia  and  related  compounds.  It  was  thus  natural  to  consider  the  flavor 
due  to  these  simple  products  of  protein  degradation.  More  recently  it 
has  been  discovered  that  the  intensity  of  flavor  does  not  necessarily  cor- 
respond to  the  content  of  the  cheese  in  these  products;  indeed  a  cheese 
may  have  a  high  content  of  nitrogen  as  ammonia  and  yet  be  low  in 
flavor. 

The  Wisconsin  Experiment  Station  has  found  that  the  volatile  fatty 
acids  of  Cheddar  cheese  increase  as  the  ripening  progresses.  In  the 
following  table  are  given  the  data  obtained  from  the  detailed  study  of  a 
normal  Cheddar  cheese. 

ACIDS  IN  ioo  G.  OF  DRY  MATTER 


3  days 

42  days 

3  months 

$M  months 

10  months 

Lactic  acid 

84  oo 

QO  28 

124  oo 

IO3    7O 

74.    IO 

Acetic  acid  

II    "\Q 

20   44 

24.    2  1 

25  86 

12    6d 

Propionic  acid 

O    4.1 

2    IS 

34.2 

I    O7 

2    6l 

Butyric  acid.  .  .  . 

O    73 

2    17 

3    (JO 

4  82 

cr    AC 

Caproic  acid  

O.OO 

o.  36 

O.o6 

I.  2<? 

2.  23 

It  will  be  noted  that  the  content  of  the  higher  volatile  acids,  those 
especially  marked  in  odor,  continually  increases.  It  is  possible  to  sepa- 
rate other  volatile  compounds  found  in  cheese  from  the  volatile  fatty 
acids  by  distilling  with  steam,  neutralizing  the  distillate  with  an  alkali 
and  redistilling;  the  second  distillate  will  contain  the  alcohols  and  esters 
present  in  the  cheese.  Such  a  distillate  prepared  from  Cheddar  cheese 
is  found  to  possess  the  characteristic  aroma  of  the  cheese  in  question. 
The  esters  it  contains  are  largely  those  of  ethyl  alcohol.  The  acid-form- 
ing bacteria,  as  stated  previously,  produce  varying  amounts  of  volatile 
acids  and  slight  amounts  of  alcohols  and  esters.  It  is  likely  that  the 
larger  part  of  the  volatile  compounds  found  in  the  ripening  cheese  is 
formed  in  fermentations  which  take  place  subsequent  to  the  initial 
fermentation  of  the  lactose.  The  flavor  of  Cheddar  cheese,  therefore, 
owes  its  origin  very  probably  to  the  fermentation  of  the  lactose,  and  to 
the  further  change  which  the  products  of  the  initial  fermentation  un- 
dergo under  the  influence  of  biological  factors  yet  unknown.  That  some 

32 


498  MICROBIOLOGY   OF   MILK   AND   MILK   PRODUCTS 

biological  factor  is  concerned  in  the  production  of  flavor  in  Cheddar 
cheese  is  indicated  by  the  fact  that  if  changes  are  made  in  the  methods 
of  manufacture,  changes  in  flavor  are  likely  to  result.  If  the  salt  is 
omitted,  the  typical  flavor  does  not  appear.  This  can  be  explained 
only  by  the  action  of  the  salt  on  certain  types  of  bacteria,  which,  in 
its  absence,  are  able  to  grow  and  produce  compounds  that  are  not 
found  in  a  normal  cheese.  Apparently  the  methods  of  manufacture 
establish  a  certain  equilibrium  in  the  bacterial  life  which  results  in  the 
production  of  definite  substances  in  amounts  varying  within  certain 
limits.  If  any  condition  is  varied  too  widely,  a  deviation  in  the  micro- 
bial  balance  is  produced  and*  the  products  formed  in  the  cheeses  are 
changed  in  kind  or  in  amounts,  either  of  which  may  result  in  a  change  of 
flavor. 

ABNORMAL  CHEESES 

The  development  of  a  normal  texture  and  flavor  in  Cheddar  cheese 
is  largely  dependent  on  the  presence  of  definite  types  of  bacteria. 
If  these  are  replaced,  wholly  or  in  part,  by  other  kinds,  the  product  is 
likely  to  suffer  in  texture,  flavor  or  both.  As  has  been  emphasized 
previously,  the  bacterial  content  of  the  milk  is  of  the  greatest  importance 
in  cheese,  since  the  organisms  in  the  milk  pass  into  the  cheese  and  there 
produce  the  same  products  as  they  would  have  done  in  the  uncurdled 
milk.  All  abnormalities  of  the  cheese  so  far  as  they  are  occasioned  by 
bacteria  are  due  to  the  abnormal  flora  of  the  milk.  To  the  raw 
material  the  maker  must  direct  his  attention  if  a  fine  product  is  to  be 
prepared. 

GASSY  CHEESE. — The  most  frequent  trouble  encountered  and  the 
one  of  greatest  economic  importance  is  the  fermentation  caused  by 
organisms  belonging  largely  to  the  B.  coli-aero genes  group.  It  has  been 
seen  that  these  produce  in  milk  gases,  such  as  carbon  dioxide  and 
hydrogen,  and  offensive  smelling  and  tasting  compounds.  In  cheese 
similar  compounds  are  formed  by  these  organisms;  the  gas  causes  the 
more  or  less  abundant  formation  of  holes  which  give  to  the  cheese  judge 
an  indication  of  what  may  be  expected  with  reference  to  flavor.  All 
milk  contains  some  of  the  gas-forming  organisms,  but  it  is  only  when 
they  are  numerous  that  marked  injury  is  done. 

Gassy  cheese  may  also  be  due  to  the  presence  of  lactose-fermenting 
yeasts  which  are  usually  found  in  milk  in  such  small  numbers  that  they 


RELATION    OF    MICROORGANISMS    TO    CHEESE  499 

cannot  compete  with  the  lactic  bacteria  in  the  fermentation  of  the  sugar 
in  the  cheese.  At  times  the  number  may  be  increased  to  such  an  extent 
that  the  major  part  of  the  sugar  is  fermented  by  them,  alcohol  and  car- 
bon dioxide  being  produced.  An  outbreak  of  gassy  Swiss  cheese  was 
found  by  Russell  and  Hastings  to  be  due  to  such  yeasts  that  had  gained 
entrance  to  the  milk  from  the  whey-barrels  because  of  careless  washing 
of  the  milk  cans.  The  cheese  makers  of  the  country  are  realizing  the 
importance  of  the  contamination  of  the  milk  from  the  transportation  of 
whey  and  milk  in  the  same  can.  The  most  practical  means  of  prevent- 
ing trouble  from  this  practice  is  to  heat  the  whey  to  68°  as  it  passes  from 
the  cheese  vat  to  the  storage  tank.  This  temperature  destroys  the 
harmful  microorganisms,  and  ft  the  storage  tank  is  kept  in  a  sanitary 
condition,  the  whey  is  sweet  when  returned  to  the  farm  in  the  milk  can. 
It  has  been  demonstrated  that  such  a  treatment  of  the  whey  results  in  a 
marked  improvement  in  the  quality  of  the  product. 

MISCELLANEOUS  ABNORMALITIES  OF  CHEESE. — Bitter  cheese  is 
produced  by  bacteria  that  form  a  bitter  principle.  An  outbreak  of 
bitter  cheese  investigated  by  Hastings  was  found  to  be  due  to  the  re- 
placement of  the  normal  acid-forming  flora  by  a  lactic  organism  which 
produced  such  an  intense  bitterness  as  to  mask  the  acid  taste  in  the 
milk  and  cheese. 

Colored  cheese  is  produced  by  chromogenic  bacteria.  In  case  the 
colonies  are  not  numerous  and  the  pigment  formed  is  not  soluble  in  any 
of  the  constituents  of  the  cheese,  the  color  will  appear  as  colored  specks, 
such  as  the  rusty  spot  investigated  by  Connel  and  Harding,  which  is  due 
to  red  forms  of  B.  rudensis.  If  the  colonies  are  very  numerous,  or  if 
the  pigment  is  soluble,  the  curd  may  be  uniformly  colored. 

Putrid  cheese  is  caused  by  the  absence  of  sufficient  acidity  to  hold 
the  putrefactive  bacteria  in  check.  This  trouble  is  rare  in  cheddar 
cheese,  since  such  cheese  is  made  from  ripened  milk.  Fruity  flavors  are 
asserted  to  be  due  to  yeasts  which  form  fruit  esters. 

Moldy  Cheese. — In  the  moist  air  of  the  curing-room  the  cheese  forms 
an  excellent  substratum  for  the  growth  of  common  molds  whose  pig- 
mented  spores  discolor  the  surface  of  the  cheese  and  thus  impair  its 
value  because  of  the  appearance  rather  than  by  any  effect  in  the  flavor. 
Cheddar  cheese  is  protected  effectively  from  molds  by  dipping  the 
cheese,  when  two  or  three  days  old,  in  melted  paraffin  which  excludes 
the  air  from  the  spores  on  the  surface  of  the  cheese. 


500  MICROBIOLOGY   OF   MILK   AND   MILK   PRODUCTS 

SPECIFIC  KINDS  OF  CHEESE 

The  greater  part  of  the  cheese  made  in  this  country  is  of  the  cheddar 
type.  Other  kinds  of  cheese  are  made,  however,  to  a  considerable 
extent.  Due  to  the  fact  that  these  cheeses  are  manufactured  primarily 
in  other  countries,  the  phrase  "  foreign  cheese "  is  often  applied  in 
contradistinction  to  the  domestic  or  cheddar  cheese.  It  has  not  been 
possible  to  manufacture  in  this  country  all  the  commercially  important 
varieties  of  foreign  cheese.  Up  to  the  present  the  manufacture  of 
certain  types  has  been  successful  only  in  the  localities  in  which  they 
originated.  Variations  in  climate  or  in  other  conditions  in  other 
localities  cause  deviations  from  the  normal  ripening.  It  is  probable 
that  when  the  knowledge  of  the  essential  biological  factors  concerned 
in  the  ripening  of  each  type  is  complete,  it  will  be  possible  to  make  any 
variety  at  will.  Considerable  progress  has  already  been  made  in  this 
country  in  the  making  of  certain  varieties  of  foreign  cheese  through  the 
work  of  the  Dairy  Division  of  the  United  States  Department 
of  Agriculture. 

CHEDDAR  CHEESE. — Cheddar  cheese,  treated  in  much  detail  in  the 
foregoing  considerations  because  it  is  the  most  important  American 
cheese,  is  made  in  England  and  her  colonies  and  in  the  United  States 


PIG.  149. — Typical  development  of  "eyes"  in  Swiss  cheese.     (Original.} 

It  appears  in  many  varieties  and  by  the  American  consumer  is  often 
called  American  cheese  in  distinction  from  the  foreign  cheeses.  This 
distinction  is  not  wholly  applicable  at  the  present  time. 

EMMENTHALER  CHEESE. — Swiss  or  Emmenthaler  cheese  originated 
in  Switzerland,  but  is  now  made  in  various  pther  countries.     A  large 


RELATION   OF   MICROORGANISMS    TO   CHEESE  501 

amount  is  made  in  Wisconsin,  Ohio  and  New  York.  It  is  characterized 
by  its  sweetish  flavor  and  by  the  so-called  "eyes,"  which  are  holes 
formed  by  gas,  produced  in  a  fermentation  that  occurs  subsequent  to 
the  fermentation  of  the  lactose  (Fig.  149).  The  number  of  eyes  is  not 
large  and  they  are  evenly  distributed  throughout  the  mass  of  the  cheese 
except  near  the  surface. 

The  cheese  is  made  from  as  fresh  milk  as  it  is  possible  to  secure. 
The  rennet  used  is  prepared  by  placing  a  piece  of  the  dried  rennet  in 
whey  and  incubating  the  same  for  twenty-four  to  thirty-six  hours  at  30°. 
This  is  employed  in  place  of  the  commercial  extract  used  by  the  Cheddar 
maker.  It  serves  not  only  to  curdle  the  milk,  but  adds  to  it  a  large 
number  of  acid-forming  bacteria  that  have  grown  in  the  rennet  solution 
during  the  period  of  incubation.  The  number  is  not,  however,  suffi- 
cient to  cause  any  development  of  acid  during  the  making  process  which 
differs  from  the  preparation  of  Cheddar  cheese  in  the  method  of  firming 
the  curd.  This  is  accomplished  by  heating  the  curd  to  52°  to"1 60°,  and 
by  cutting  it  into  pieces  scarcely  larger  than  grains  of  wheat.  The  salt 
is  applied  to  the  exterior  of  the  cheese  by  immersion  in  brine  for  one  to 
four  days  and  by  sprinkling  salt  on  the  surface. 

The  fermentation  of  the  lactose  proceeds  rapidly  during  the  pressing 
and  subsequent  thereto,  so  that  within  a  few  days  the  sugar  has  disap- 
peared. The  lack  of  the  development  of  acid  during  the  making  probably 
results  in  a  somewhat  different  relation  between  the  acid  and  protein 
from  that  existing  in  a  Cheddar  cheese,  which,  together  with  the  ab- 
sence of  salt  gives  a  somewhat  different  environment,  thus  making  possi- 
ble the  development  of  a  different  flora.  There  is  no  ground  for  believing 
that  the  agents  concerned  in  the  proteoly  tic  changes  are  other  than  those 
that  function  in  Cheddar  cheese.  The  flavor  must,  however,  be  due  to 
other  factors;  this  is  indicated  by  the  fact  that  if  the  milk  is  ripened 
as  in  the  Cheddar  process,  or  if  salt  is  added  to  the  curd  the  flavor  will 
approximate  the  Cheddar  flavor.  The  formation  of  the  eyes  is  inhib- 
ited by  salt,  as  is  indicated  by  their  relative  scarcity  in  the  outer  layers 
of  the  cheese.  Jensen  has  shown  that  the  eyes  are  due  to  the  fermenta- 
tion of  lactates  with  the  formation  of  propionic  and  acetic  acids,  and 
carbon  dioxide.  The  causal  organism  is  found  in  the  milk  and  the  whey 
rennet.  It  is  believed  that  lactic  bacteria  of  the  Bad.  bulgaricum  group 
are  important  factors  in  the  ripening  of  Swiss  cheese.  They  are  pre- 
sent in  large  numbers  in  the  rennet  and  cheese.  Mixed  cultures  of  an 


502  MICROBIOLOGY    OF   MILK   AND    MILK   PRODUCTS 

organism  of  this  group  and  a  mycoderma  are  used  with  success  in 
Switzerland  for  the  inoculation  of  the  whey  in  which  the  rennet  is  to  be 
soaked.  The  exact  role  of  this  form  of  lactic  organism  is  not  known;  de 
Freudenreich  considered  them  to  be  concerned  in  the  proteolysis  of 
the  paracasein,  since  he  had  found  that  the  content  of  sterile  milk  in 
soluble  nitrogen  increased  when  inoculated  with  the  organism.  It  has 
been  found  possible  by  Rogers  and  his  associates  to  employ  commercial 
rennet  and  pure  cultures  of  organisms  of  the  Bact.  bulgaricum  group 
in  the  making  of  Swiss  cheese.  It  has  also  been  found  advantageous  to 
add  to  the  milk  a  small  amount  of  a  culture  of  the  eye-forming  organ- 
ism. It  seems  probable  that  the  use  of  these  pure  cultures  will  result 
in  a  greater  uniformity  of  the  product  than  it  has  been  possible  to 
attain  by  following  the  empirical  methods  commonly  used.  It  is 
probable  that  the  formation  of  eyes  and  the  flavoring  compounds  are 
due,  in  part  at  least,  to  the  same  factors. 

In  the  other  kinds  of  cheeses  to  be  described,  the  r61e  of  the  acid- 
forming  bacteria  is  similar,  if  not  identical,  to  their  r61e  in  Cheddar 
cheese,  i.e.,  in  activating  the  pepsin  of  the  rennet  and  in  preventing 
the  growth  of  putrefactive  bacteria.  The  factors  concerned  in  flavor 
development  are  different. 

ROQUEFORT  CHEESE. — This  cheese,  which  is  prepared  almost 
exclusively  in  the  Department  of  Aveyron  in  southern  France,  is  made 
from  sheep's  milk.  Its  most  striking  characteristic  is  the  marbled 
or  mottled  appearance  of  the  interior,  due  to  the  growth  of  a  mold, 
Penicillium  roqueforti,  Thorn.  The  curd  is  inoculated  with  the  mold, 
when  it  is  placed  in  the  press,  by  sprinkling  the  curd  with  bread  crumbs 
on  which  the  mold  has  grown.  The  growth  and  sporulation  of  the 
mold  in  the  interior  of  the  cheese  are  favored  by  piercing  it  with 
small  needles,  thus  admitting  air.  The  characteristic  flavor  is  due, 
partially  at  least,  to  the  mold. 

This  cheese  is  cured  in  caves  having  a  temperature  below  15°. 
The  fermentative  processes  are  apparently  closely  dependent  on  the 
moisture  and  temperature  conditions  of  the  curing  room.  This 
emphasizes  the  importance  of  biological  factors  in  the  ripening  process. 

GORGONZOLA  CHEESE,  prepared  in  Italy  from  cow's  milk,  and 
STILTON  CHEESE,  made  in  England  are  similar  to  Roquefort  in 
appearance  and  contain  the  same  mold — Penicillium  roqueforti. 

CAMEMBERT  CHEESE. — The  soft  cheeses  are  best  represented  by 


RELATION   OF   MICROORGANISMS   TO   CHEESE  503 

this  important  French  cheese  made  from  cow's  milk  by  the  addition 
of  rennet.  The  milk  is  ripened  to  an  acidity  of  0.20  to  0.25  per  cent 
before  the  addition  of  the  rennet.  The  curd,  which  thus  contains 
many  acid-forming  bacteria,  is  neither  cut  nor  heated  so  that  the 
maximum  amount  of  whey  is  retained.  The  curd  is  placed  in  small 
hoops  and  allowed  to  drain  without  pressure.  Salt  is  applied  to  the 
surface  of  the  cheese. 

The  milk  sugar  is  rapidly  fermented  and  the  resulting  acidity 
is  high,  for  the  cheese  contains  60  to  70  per  cent  of  moisture  when 
fresh  and  50  per  cent  when  ready  for  consumption.  The  high  moisture 
content  of  the  cheese  and  the  humidity  and  temperature  conditions 
of  the  curing  room  favor  the  rapid  development  of  microorganisms 
on  the  surface  of  the  cheese.  Both  molds  and  bacteria  thrive  under 
the  influence  of  these  favorable  conditions,  changing  the  cheese  to 
a  soft,  smooth  and  butter-like  mass,  while  a  characteristic  flavor  is 
developed. 

In  three  or  four  days  the  cheese  becomes  covered  with  the  growth 
of  Oidium  lactis;  the  characteristic  mold  of  Camembert  cheese,  Peni- 
cillium  camemberti,  appears  later,  within  five  to  six  days.  These 
molds  reduce  the  acidity  of  the  curd,  and  through  the  enzymes,  which 
they  produce  and  which  gradually  diffuse  into  the  cheese,  proteolyze  the 
curd  very  completely.  The  appearance  of  the  cheese  when  cut  in- 
dicates the  depth  to  which  the  enzymes  have  penetrated;  when  the 
entire  mass  is  acted  upon,  the  cheese  is  ready  for  use.  The  reduction  of 
the  acidity  by  the  molds  exposes  the  cheese  to  the  attacks  of  putre- 
factive bacteria  and  it  soon  becomes  unfit  for  use  after  it  is  completely 
ripened.  Several  kinds  of  bacteria  are  found  in  the  slimy  surface 
layer,  but  their  role  is  not  known. 

The  development  of  the  characteristic  flavor  and  aroma  is  dependent 
on  a  certain  relation  between  the  various  biological  agents  concerned 
in  the  ripening.  This  balance  is  dependent  on  very  narrow  conditions 
of  temperature  and  humidity;  slight  changes  in  these  environmental 
conditions  favor  or  retard  the  individual  types  in  varying  degrees.  If 
the  equilibrium  essential  for  the  development  of  typical  flavor  is 
destroyed,  this  cheese  fails  to  ripen  properly  and  is  of  low  value.  The 
manufacture  of  Camembert  cheese  is  a  delicate  problem  in  the  ecology 
of  microorganisms,  and  because  of  this  fact  the  manufacture  is  attended 
with  greater  difficulties  than  is  the  case  with  most  types  of  hard  cheese. 


CHAPTER  IV* 

RELATION  OF  MICROORGANISMS  TO  SOME  SPECIAL 
DAIRY  PRODUCTS 

GENERAL 

There  is  a  number  of  special  dairy  products  which  do  not  normally 
come  into  a  discussion  of  market  milk,  butter  or  cheese,  but  which  are 
of  considerable  importance.  A  book  of  this  sort  would  not  be  complete 
without  a  discussion  of  some  of  these  products  from  the  bacteriological 
standpoint.  Some  of  these  special  products  have  been  developed 
as  commercial  enterprises  and  the  processes  of  manufacture  have  been 
zealously  guarded  as  trade  secrets.  The  result  is  that  there  is  very  little 
available  data  on  the  manufacture  of  these  products  and  very  little 
authoritative  knowledge  about  their  bacteriological  condition.  It  is, 
therefore,  difficult  to  give  a  full  discussion  of  the  microbiology  of  these 
products.  A  few  of  the  more  impqrtant  ones  will  be  discussed,  however. 

CONDENSED  MILK 

There  are  at  least  three  quite  distinct  kinds  of  condensed  milk 
made  under  conditions  which  result  in  an  entirely  different  bacteriolog- 
ical condition  in  the  finished  product.  These  different  products  must, 
therefore,  be  considered  separately.  Condensed  milk  means  simply 
milk  from  which  a  large  part  of  the  water  has  been  removed,  thus 
decreasing  its  bulk,  the  purpose  being  to  lessen  the  cost  of  transportation 
and  to  increase  the  keeping  quality  of  the  product.  Water  is  removed 
from  milk  by  some  process  of  heating,  either  with  or  without  vacuum, 
the  heating  process  being  more  or  less  equivalent  to  pasteurization. 

SWEETENED  CONDENSED  MILK. — This  product  is  made  by  reducing 
cow's  milk  at  the  ratio  of  two  and  one-half  to  two  and  three-fourths 
parts  of  fresh  milk  to  one  part  condensed  milk,  by  means  of  heat 
and  the  addition  of  cane  sugar.  It  is  then  put  up  in  sealed  cans. 

*  Prepared  by  W.  A.  Stocking. 

504 


RELATION  OF  MICROORGANISMS  TO  SPECIAL  DAIRY  PRODUCTS       505 

It  is  not  intended  to  be  sterile.  The  degree  of  heat  to  which  it 
is  subjected  is  not  sufficient  to  kill  all  of  the  microorganisms 
present  and  it  is  also  subject  to  infection  after  the  condensing 
is  completed.  Cane  sugar  is  added  to  the  milk,  making  the  final 
product  contain  about  25  per  cent  of  water,  35  per  cent  milk  solids 
and  40  per  cent  cane  sugar.  The  low  percentage  of  moisture  together 
with  the  added  sugar  tends  to  preserve  this  product  against  the  action 
of  microorganisms.  There  may  be  some  bacterial  growth,  the 
rapidity  depending  upon  the  temperature  at  which  the  product  is 
kept,  but  it  is  usually  slow  and  milk  prepared  in  this  way  will  keep 
for  a  considerable  time  without  undergoing  marked  bacterial  changes. 
When  gas  producing  bacteria  exist  in  the  milk  and  the  cans  containing 
the  organisms  are  allowed  to  remain  at  warm  temperatures,  they  will 
develop  in  spite  of  the  large  percentage  of  sugar,  producing  suffi- 
cient amounts  of  gas  to  cause  the  ends  of  the  cans  to  bulge  out.  Such 
cans  are  known  commercially  as  "swell-heads." 

UNSWEETENED  CONDENSED  OR  EVAPORATED  MILK. — In  this  form  of 
condensed  milk  approximately  the  same  amount  of  moisture  is  removed 
as  in  the  sweetened  product  but  no  sugar  is  added.  The  decreased 
amount  of  moisture  tends  to  prevent  the  rapid  growth  of  bacteria,  but 
this  is  not  enough  to  guarantee  the  keeping  quality  of  the  product. 
After  the  milk  is  condensed  it  is  put  into  cans,  hermetically  sealed, 
and  then  placed  in  steam  sterilizers  and  subjected  to  temperatures  some- 
what above  the  boiling-point.  In  this  way  the  milk  is  heated  a  suffi- 
cient length  of  time  to  insure  perfect  sterilization  of  the  contents  of  the 
cans.  If  this  process  is  properly  done,  the  finished  product  contains  no 
living  microorganisms  and  from  the  bacteriological  standpoint  the  milk 
should  keep  indefinitely. 

Sometimes  the  unsweetened  product  is  sold  in  bulk  in  cans.  In 
this  case  it  is  subject  to  more  or  less  contamination  after  heating  and  is 
not  sterile,  but  because  of  the  small  amount  of  moisture  and  the  concen- 
tration of  the  milk  solids,  the  bacteria  do  not  develop  rapidly  and  if 
kept  at  a  cool  temperature,  the  milk  will  keep  for  some  time  without 
undergoing  appreciable  biological  fermentations. 

CONCENTRATED  MILK. — There  is  now  on  the  market  a  form  of  con- 
densed milk  prepared  by  a  different  process,  which  is  commonly  known 
as  concentrated  milk.  By  this  method  the  water  in  the  milk  is  removed 
by  means  of  dry  air  instead  of  by  vacuum  as  is  the  case  with  condensed 


5o6 


MICROBIOLOGY   OF   MILK  AND   MILK   PRODUCTS 


milk.  The  milk  is  first  heated  and  then  air  under  pressure  is  forced 
through  it.  By  this  process  the  milk  is  heated  to  a  temperature  of 
60°  (i4o°F.),  and  this  temperature  maintained  for  two  hours,  during 
which  time  air  is  forced  through  the  milk  causing  violent  agitation  and 
the  removal  of  the  moisture.  At  the  end  of  this  time  the  milk  is  re- 
duced to  one-fourth  its  original  volume.*  The  result  of  this  process  is  a 
pasteurized  milk,  with  a  marked  reduction  of  the  original  germ  content. 
Investigations  by  Conn  failed  to  show  the  presence  of  B.  coli  in  milk 
prepared  by  this  process.  The  reduction  in  the  bacterial  content  of  the 
milk  is  similar  to  that  secured  by  other  methods  of  pasteurization.  No 
additional  sugar  is  added  to  this  milk  so  the  product  is,  therefore,  a  pas- 
teurized milk  containing  a  small  amount  of  moisture.  Because  of  the 
small  amount  of  moisture  and  the  concentration  of  the  milk  solids,  the 
bacteria  which  survive  the  heating  process  do  not  grow  rapidly  at  low 
temperatures.  The  following  figures  will  serve  to  illustrate  the  effect  of 
this  process  upon  the  bacterial  content  of  milk:  - 


Bacterial  count  per  c.c.  in  original  milk 


Bacterial  count  per  c.c.  in  finished 
product 


1,250,000 
3,000,000 
518,000 
894,000 
796,000 
150,000 


15,000 

21,000 

26,000 

9,950 

10,000 

5,000 


The  rate  at  which  the  bacteria  develop  in  this  milk  is  shown  by  the 
following  counts: 


Bacterial  count  per  c.c. 


2  days  old 

4  days  old 

6.  days  old 

I 

2 

18,000 
SS.ooo 

]        • 
39,000 

28,000 

46,000 
39,000 

3 
4 

3,500 
4,400 

11,000 

5,270 

10,000 
4,630 

The  lack  of  moisture  and  concentration  of  milk  solids  prevents  the 
rapid  growth  of  these  organisms  so  that  bacterial  changes  do  not  take 

•  Data  furnished  by  H.  W.  Conn. 


RELATION  OF  MICROORGANISMS  TO  SPECIAL  DAIRY  PRODUCTS      507 

place  as  rapidly  as  in  ordinarily  pasteurized  milk  retaining  its  normal 
moisture. 

POWDERED  MILK. — This  product  is  produced  by  carrying  the  extrac- 
tion of  the  water  farther  than  in  the  case  of  the  condensed  milks.  The 
water  is  removed  to  a  point  where  the  milk  solids  can  be  reduced  to  a 
powdered  form.  This  product  contains  the  original  milk  solids  with  a 
very  small  percentage  of  moisture  usually  not  more  than  2%  per  cent. 
There  are  several  forms  of  powdered  milk  now  on  the  market  produced 
by  somewhat  different  methods.  In  some  cases  the  moisture  is  removed 
from  the  milk  by  its  being  exposed  to  a  heated  surface  in  a  thin  layer. 
Sometimes  the  drying  is  done  in  vacuum.  The  resulting  product  is  dry 
and  can  be  ground  to  the  form  of  flour. 

Another  process  is  to  remove  the  moisture  by  spraying  the  milk  by 
means  of  an  atomizer  into  the  top  of  a  hot  chamber,  the  moisture  being 
removed  while  the  fine  particles  of  milk  are  falling  to  the  floor.  By 
this  process  the  product  accumulates  on  the  floor  as- a  very  dry  flour  and 
does  not  require  any  grinding.  In  the  first  process  the  heat  is  sufficient 
to  pasteurize  the  milk  while  in  the  latter  process  it  is  pasteurized  before 
being  subjected  to  the  drying  process.  The  powdered  milks  do  not 
claim  to  be  sterile  but  are  preserved  against  subsequent  action  of  micro- 
organisms because  of  the  very  low  percentage  of  moisture  which  they 
contain.  It  is  probable  that  there  is  no  appreciable  increase  in  the 
number  of  bacteria  in  milk  flour  and  the  product  will  keep  for  a  long  time 
without  undergoing  bacterial  fermentations. 

CANNED  BUTTER  AND  CHEESE 

Some  effort  has  been  made  to  put  up  butter  and  cheese  in  hermet- 
ically sealed  cans,  the  purpose  being  to  increase  the  keeping  qualities  of 
the  products  and  influence  the  flavor  by  controlling  the  development  of 
the  aerobic  bacteria.  Only  a  limited  amount  of  bacteriological  work 
has  been  done  on  these  canned  products  and  the  biological  changes 
which  take  place  in  them  are  not  very  well  known. 

SPECIAL  MILK  DRINKS  MADE  BY  THE  ACTION  OP  MICROORGANISMS 

From  time  immemorial  fermented  or  sour  milk  has  been  used  as  an 
article  of  food.  We  are  told  that  Abraham*  placed  "curdled  milk" 

•  Genesis  18:8.  The  Hebrew  word  "hemah"  translated  in  the  English  authorized  version 
of  the  Bible  "butter"  means  "curdled  milk."  Century  Bible,  Vol.  Judges  and  Ruth,  p.  ?a. 


508  MICROBIOLOGY   OF   MILK   AND   MILK   PRODUCTS 

before  his  guests  and  that  Moses  told  the  Israelites  that  curdled  milk 
was  one  of  the  blessings  which  Jehovah  had  given  .to  his  chosen  people.* 
History  also  tells  us  that  the  wandering  tribes  of  Arabia  used  fermented 
milk  as  a  beverage.  For  centuries  many  of  the  tribes  of  eastern  Europe 
and  western  and  middle  Asia  and  parts  of  Africa  have  used  sour  milk 
for  food.  Each  of  these  regions  appears  to  have  had  its  own  particular 
milk  beverage  resulting  from  the  particular  bacterial  flora  of  the  region. 

The  sour  milk  products  which  are  now  on  the  market  under  a 
variety  of  names  have  been  derived  from  these  original  sour-milk 
drinks  of  antiquity.  Fermented  milk  beverages  have  become  very 
popular  during  the  last  few  years  among  all  the  civilized  peoples, 
partly  because  they  make  a  pleasant  drink  but  more  especially  because 
of  their  supposed  therapeutic  value,  f 

KUMYSS  (KOUMISS,  KUMISS,  ETC.). — Kumyss  derives  its  name  from 
the  Kumanes,  a  Russian  tribe  which  lived  along  the  river  Kuma. 
This  drink  was  prepared  from  mare's  milk  by  placing  it  in  a  leather 
bag  and  adding  a  small  amount  of  old  kumyss  as  a  starter.  J  In  this 
country  kumyss  is  made  from  cow's  milk.  This  product  is  now  placed 
upon  the  market  by  a  number  of  companies  who  keep  their  methods, 
so  far  as  possible,  from  their  rivals  by  maintaining  strict  secrecy  in 
regard  to  the  methods  of  preparation.  Dr.  Piffard||  who  has  done 
special  work  on  this  product  states  that  kumyss  is  fermented  by  the 
action  of  yeasts  and  lactic  bacteria.  This  fermentation  produces 
approximately  i  per  cent  of  alcohol  and  about  0.75  per  cent  of  acid. 
Kumyss  is  strongly  effervescent.  The  lactic  organisms  used  in  the 
preparation  of  this  material  appear  to  be  a  strain  of  the  common  Bact. 
lactis  acidi. 

Kumyss  can  be  easily  prepared  in  the  household  by  the  addition 
of  cane  sugar  and  baker's  yeast  to  fresh,  warm  milk  which  should  be 
kept  at  a  temperature  of  about  38°  (ioo°F.)  until  gas  begins  to  form. 
It  should  then  be  bottled  and  be  kept  at  a  cool  temperature.  In  one 
or  two  days  a  slight  amount  of  alcohol  will  be  formed  and  a  sufficient 
amount  of  carbon  dioxide  to  cause  marked  effervescence. 

KEFIR  (KEFYR,  KEPHIR,  KEFR,  ETC.). — Kefir  was  originally  made 
and  used  by  the  inhabitants  of  the  Caucasus  Mountains.  It  was 

*  Deut.  32:14. 

f  Metchnikoff's  Prolongation  of  Life. 

j  Milch  Zeitung,  September,  1889. 

U  New  York  Medical  Journal,  January  4,  1908. 


RELATION  OF  MICROORGANISMS  TO  SPECIAL  DAIRY  PRODUCTS      509 

made  from  the  milk  of  goats,  sheep  or  cows  and  was  fermented  by  the 
addition  of  "kefir  grains"  to  the  milk.  The  origin  of  these  kefir  grains 
is  unknown  but  the  natives  believe  that  they  were  the  gift  of  Mahomet 
and  are  carefully  preserved  by  them. 

Kefir  was  prepared  by  the  natives  by  placing  milk  in  a  goat-skin 
bag  and  shaking  it  at  intervals  until  it  began  to  ferment.  The  kefir 
grains  were  then  removed,  dried  and  preserved  for  future  use.  The 
fermented  kefir  was  also  used  as  a  starter  for  inoculating  new  lots. 
This  beverage  is  now  commonly  made  by  more  scientific  methods.* 
The  principal  points  to  be  observed  in  the  preparation  of  kefir  are 
cleanliness  and  proper  temperature  for  fermentation  and  the  regulation 


FIG.  150. — A  large-sized  kefir  grain  and  the  three  species  of  bacteria  of  which  it  is 
composed.     (From  Conn,  after  de  Freudenreich.) 

of  the  fermentation  so  that  not  the  acid  but  the  alcoholic  fermentation 
will  prevail,  f  Good  kefir  should  be  highly  effervescent,  should  be  free 
from  lumps  and  contain  about  i  per  cent,  of  acid  but  show  no  marked 
tendency  to  whey  off.  According  to  Kern,  kefir  is  fermented  by  a 
mixed  culture  of  yeasts  and  bacteria  in  symbiosis.  He  found  but  one 
form  of  bacteria  present  in  the  cultures  he  studied.  De  Freuden- 
reichj  made  an  extended  study  of  the  flora  of  kefir.  He  prepared 
the  kefir  from  the  kefir  grains  and  isolated  the  organisms  present, 
putting  these  organisms  together  in  different  combinations  in  order 
to  determine  which  were  necessary  for  the  proper  fermentation  of 
the  kefir.  He  found  the  kefir  contained  four  different  organisms: 

*  Milch  Zeitung,  1885,  p.  209. 

t  P.  Stohman,  Milch  and  Molkerei  Products,  p.  1006  to  1013. 

j  Centr.  fur  Bakt.  Abt.  2,  Vol.  3,  189?. 


510  MICROBIOLOGY   OF   MILK  AND   MILK  PRODUCTS 

yeasts,  streptococci,  micrococci,  and  bacilli.  The  yeasts  and  strepto- 
cocci were  plated  in  gelatin  without  difficulty  but  it  was  very  difficult 
to  grow  the  other  two  organisms  present  on  any  artificial  media. 
He  concluded  that  the  yeasts  present  in  kefir  are  not  identical  with 
the  species  commonly  used  in  making  beer  and  named  it  Sacchar- 
omyces  kefir.  The  streptococcus  curdled  milk  in  less  than  forty-eight 
hours  at  a  temperature  of  37°  but  the  micrococcus  did  not  curdle 
milk  at  all,  although  it  produced  a  considerable  amount  of  acid. 

De  Freudenreich  changed  the  name  of  the  bacillus  from  Dispora  caucasica, 
given  it  by  Kern  to  B.  caucasicus,  because  it  did  not  produce  spores  as  Kern  sup- 
posed. He  also  found  that  this  organism  would  not  grow  at  all  on  media  without 
sugar,  very  slightly  on  milk,  serum,  agar,  and  best  of  all  in  milk,  in  which  it  produces 
both  gas  and  acid  without  curdling  the  milk.  This  organism  is  5/z  or  6/i  in  length 
by  if*  in  width,  is  slightly  motile  and  retains  Gram's  stain.  It  has  a  thermal  death- 
point  of  55°  for  five  minutes. 

The  preparation  of  good  kefir  seems  to  depend  upon  the  combined 
action  of  the  four  types  of  organisms  described.  Kefir  is  sometimes 
prepared  without  the  use  of  the  kefir  grains*  by  placing  milk  in  bottles 
to  which  is  added  a  small  amount  of  compressed  yeast  and  sucrose. 
The  bottles  are  then  held  at  a  temperature  of  10°  to  15°  about  fifteen 
hours  and  shaken  occasionally.  Kefir  prepared  in  this  way  gives  an 
effervescent  mild  flavored  drink. 

LEBEN. — For  centuries  the  Egyptians  have  used  a  fermented  milk 
drink  known  as  leben  or  leben  raib.  This  was  prepared  from  the  milk 
of  cows,  buffaloes,  and  goats.  In  general  it  resembles  the  other  fer- 
mented milk  drinks  in  the  fact  that  the  fermentation  is  produced 
by  yeasts  and  a  variety  of  other  microorganisms  working  together. 
At  least  one  yeast  and  three  species  of  bacteria  seem  to  be  normal 
to  this  product.  A  fermented  milk  drink  very  similar  to  leben  is 
also  used  in  Algeria.  The  exact  action  of  each  microorganism  concerned 
in  the  fermentation  of  this  product  is  not  certain,  but  it  is  probable 
that  all  of  the  species  are  essential  for  the  production  of  the  particular 
flavor  and  consistency  of  the  fermented  product.  It  is  claimed  that 
the  fermentation  that  takes  place  in  the  milk  renders  it  more  digest- 
ible than  raw  milk.  For  this  reason  it  is  recommended  for  the  use 
of  invalids  and  persons  having  weak  digestion. 

YAHOURTH  OR  MATZOON  (YOGURT,  YAHOURD,  MADZOON,  ETC.). — A 
fermented  milk  drink  known  by  one  of  the  above  names  has  been  used 

*  Milch  Zeitung,  1888,  p.  393. 


RELATION  OF  MICROORGANISMS  TO  SPECIAL  DAIRY  PRODUCTS      $11 

by  the  Bulgarian  tribes  for  a  long  time.  Some  years  ago  it  was  studied 
and  brought  to  public  notice  by  the  investigations  and  writings  of 
Metchnikoff,*  who  was  struck  by  the  longevity  of  the  tribes  using  this 
product  as  a  part  of  their  regular  diet.  As  a  result  of  his  investigations, 
Metchnikoff  has  advanced  his  theory  regarding  .the  antiseptic  power 
of  certain  strains  of  lactic  bacteria  in  the  digestive  tract.  His  theory 
is  that  certain  species  or  types  of  bacteria  which  are  able  to  resist 
the  action  of  the  stomach  and  can,  therefore,  pass  through  into  the 
intestines  have  the  power  of  checking  the  growth  of  the  putrefactive 
bacteria  existing  there  and  thereby  prevent  the  production  and  ab- 
sorption of  bacterial  toxins  which  cause  autointoxication.  As  a  result 
of  his  experiments,  Metchnikoff  came  to  the  conclusion  that  the  acid 
organism  (Bad.  bulgaricum)^  found  in  yahourth  was  able  to  establish 
itself  in  the  intestinal  tract  and  produce  enough  lactic  acid  to  hold 
in  check  the  putrefactive  processes  which  otherwise  exist  there. 

Yahourth  is  made  by  the  Bulgarians  in  skin  bags  in  the  same  way 
that  the  Russian  tribes  prepare  kumyss.  It  is  similar  to  the  other  fer- 
mented drinks  already  described  in  the  fact  that  it  is  produced  by  a 
mixed  flora  of  microorganisms.  At  least  one  yeast  is  present  and  two 
or  more  species  of  bacilli  capable  of  producing  lactic  acid  in  relatively 
large  amounts.  These  two  organisms  are  known  as  Bact.  bulgaricum 
and  Bacillus  paralacticus.  Herter  states  that  Bact.  bulgaricum  is  4ju  to 
6/i  in  length. by  iju  in  width  and  grows  singly  or  in  pairs  and  occasionally 
in  chains.  It  stains  with  ordinary  aniline  dyes  and  by  Gram's  method. 
It  grows  with  difficulty  on  ordinary  laboratory  media  and  is  therefore 
hard  to  obtain  in  pure  cultures.  These  organisms  produce  a  much 
higher  percentage  of  acid  than  the  common  Bact.  lactis  acidi  and  also 
grow  at  a  much  higher  temperature. 

This  makes  it  possible  to  secure  it  in  practically  pure  cultures  by 
growing  it  in  milk  at  a  high  temperature.  Grown  in  pure  cultures,  the 
Bact.  bulgaricum  will  produce  from  i  to  2  or  more  per  cent  of  acidity. 
It  grows  well  at  temperatures  between  37°  and  40°  and  even  higher. 
Recently  a  number  of  fermented  milk  drinks  have  been  put  upon  the 
market  which  have  evidently  been  derived  from  the  yahourth.  These 
are  sold  under  such  trade  names  as  zoolak,  vitalac,  yogurt,  fermenlactyl, 
etc.  The  flora  of  these  preparations  appears  to  be  practically  the  same 
as  that  of  the  original  yahourth. 

*  El.,  Metchnikoff,  Prolongation  of  Life. 

f  Hastings  has  found  this  organism  also  common  in  cow's  milk  in  this  country. 


512  .MICROBIOLOGY    OF   MILK   AND    MILK   PRODUCTS 

All  of  the  fermented  milk  drinks  thus  far  discussed  are  similar  in  that 
each  contains  a  variety  of  microorganisms,  made  up  of  at  least  one 
species  of  yeast  with  one  or  more  species  of  bacteria,  capable  of  produc- 
ing greater  or  less  amounts  of  acid.  In  some,  as  in  the  case  of  kefir,  the 
yeast  fermentation  is  allowed  to  predominate,  while  in  others,  like  ya- 
hourth,  the  action  of  the  yeasts  is  held  in  check  by  the  rapid  develop- 
ment of  the  acid  by  the  Bact.  bulgaricum.  All  of  these  drinks  are  com- 
monly recommended  by  physicians  because  of  their  beneficial  effect 
upon  the  digestive  tract. 

ARTIFICIAL  BUTTERMILK. — In  recent  years  there  has  developed  an 
important  industry  in  the  manufacture  of  artificial  buttermilk.  This 
is  usually  made  by  inoculating  skim-milk  with  a  culture  of  lactic 
bacteria,  either  Bact.  lactis  acidi,  or  Bad.  bulgaricum  or  a  combination 
of  these  two  types.  In  making  the  artificial  buttermilk,  yeasts  are  not 
commonly  added.  After  the  milk  becomes  coagulated,  it  is  then 
churned  in  order  to  give  it  a  smooth,  creamy  consistency,  after  which 
it  may  be  bottled  and  kept  for  some  time  by  holding  at  low  tempera- 
tures. Sometimes  a  small  percentage  of  whole  milk  is  added  at  the  time 
of  churning  to  make  the  finished  product  more  closely  resemble  natural 
buttermilk.  In  making  artificial  buttermilk,  the  skim-milk  is  fre- 
quently pasteurized  in  order  to  get  rid  of  the  miscellaneous  flora  which 
it  contains.  The  finished  product,  therefore,  differs  from  ordinary  but- 
termilk in  the  fact  that  it  contains  nearly  pure  cultures  of  the  lactic 
organisms  while  the  natural  buttermilk  will  contain  a  more  or  less 
miscellaneous  flora  in  which  the  acid  organisms  predominate.  It  is 
possible  to  obtain  a  more  uniform  product  in  the  artificial  buttermilk 
than  in  the  natural  product,  and  this  is  perhaps  responsible  for  the 
rapid  development  of  this  industry.  All  of  these  fermented  milk 
drinks  contain  enormous  numbers  of  microorganisms,  usually  millions 
per  c.c. 

ICE  CREAM 

Ice  cream  is  one  of  the  important  manufactured  dairy  products  and 
its  use  seems  to  be  increasing  steadily.  Its  bacterial  flora  varies  with 
the  materials  used  in  its  manufacture  and  the  conditions  under  which  it 
is  made.  It  may  be  made  from  fresh  cream  which  is  only  a  few  hours 
old  and  under  good  sanitary  conditions.  On  the  other  hand,  it  may  be 
made  from  cream  which  has  been  produced  and  handled  under  unsani- 
tary conditions,  kept  in  storage  for  a  number  of  days  and  finally  manu- 


RELATION  OF  MICROORGANISMS  TO  SPECIAL  DAIRY  PRODUCTS       513 

factured  in  surroundings  not  conducive  to  a  low  bacterial  content.  We 
are  not  surprised,  therefore,  to  find  a  very  wide  variation  in  the  germ 
content  of  ice  cream,  as  it  is  placed  upon  the  market. 

An  examination  of  263  samples  of  ice  cream  collected  in  the  city  of 
Washington*  showed  an  average  germ  content  of  over  26,600,000  per 
c.c.  The  lowest  count  obtained  was  37,500  and  the  maximum  was 
365,000,000.  A  similar  study  of  commercial  ice  cream  in  Philadelphia f 
showed  the  average  bacterial  content  to  be  very  high.  The  lowest 
count  found  was  50,000  per  c.c.,  while  the  highest  count  was  150,200,000. 
In  this  work  it  was  found  that  the  bacterial  content  of  the  ice  cream  was 
in  quite  direct  relation  to  the  sanitary  conditions  of  the  establishment 
where  the  ice  cream  was  manufactured.  When  ice  cream  is  manufac- 
tured in  a  city  from  materials  which  have  been  shipped  in  from  consider- 
able distances  and  frequently  held  for  several  days  in  cold  storage,  it  is 
not  surprising  that  the  germ  content  of  the  manufactured  product 
should  be  high.  In  some  establishments  the  cream  is  pasteurized  be- 
fore manufacturing,  while  in  others  it  is  used  in  its  raw  condition. 
Under  present  commercial  conditions  considerable  amounts  of  con- 
densed milk,  and  frequently  unsalted  butter,  are  used  for  ice 
cream  making.  EllenbergerJ  found  that  while  all  the  ingredients 
used  contained  some  bacteria  by  far  the  greater  numbers  were  in  the 
cream  and  condensed  milk.  This  is  shown  by  the  following  table  giv- 
ing the  average  plate  counts  from  the  ingredients  used. 

AVERAGE  PLATE  COUNTS  OF  INGREDIENTS 

Maximum  count 


Standardized  cream. 

I    ICQ 

37  6oo,OOO 

Condensed  milk  

3I,«COO 

<?o,  800,000 

Sugar     .  . 

20 

2CC 

Gelatin  

48 

80  1 

Flavoring  

10 

721 

In  normal  cream  held  for  some  time,  the  lactic  bacteria  should  exist 
in  considerable  numbers,  but  when  cream  is  held  at  low  temperatures 
these  organisms  do  not  develop  rapidly.  Pennington  found  that  cer- 
tain species  of  streptococci  developed  quite  rapidly  in  cream  held  at 


*  Results  of  work  done  under  the  direction  of  G.  W.  Stiles, 
t  Work  done  under  the  direction  of  Dr.  M.  E.  Pennington, 
J  Cornell  Memoir  No.  18, 
33 


MICROBIOLOGY   OF   MILK   AND   MILK  PRODUCTS 


refrigerator  temperatures.  Streptococci  were  found  in  fifty-five  (80  per 
cent)  of  the  sixty-eight  samples  examined.  It  was  found  that  at  refrig- 
erator temperatures  the  relative  growth  of  these  organisms  was  greater 
than  at  higher  temperatures,  a  fact  which  may  account,  in  part  at  least, 
for  the  frequency  with  which  these  organisms  occur  in  ice  cream. 

Frequently  ice  cream  is  held  for  a  considerable  time  in  a  frozen  con- 
dition before  it  is  sold.  It  has  generally  been  supposed  that  there  is  no 
bacterial  growth  in  material  which  is  held  below  the  freezing  tempera- 
ture. This,  however,  did  not  seem  to  be  the  case  in  samples  examined 
by  the  investigators  already  mentioned.  They  found  in  samples  held 
about  a  month  that  there  was  normally  a  decrease  in  the  bacterial  count 
and  also  in  the  amount  of  gas  production  for  a  number  of  days,  after 
which  there  was  frequently  a  marked  increase  in  the  bacterial  counts. 
These  results  would  seem  to  indicate  that  even  in  the  frozen  condition 
there  may  be  some  increase  in  the  number  of  bacteria  present.  Ellen- 
berger*  found  the  same  general  conditions  as  these  earlier  investigators 
as  shown  by  the  following  chart. 


Per  cent  of 

bacteria 

150 


80 
70 

60 

ad 

40 

30 

Days  in 
storage 


\ 


10 


20         30 


40 


50 


60        70 


80 


90 


100 


120 


FIG.  151. — Average  percentage  increase  and  decrease  of  bacteria  in  fourteen  sam- 
ples of  ice  cream  during  a  storage  period  of  i2oMays. 


*  Cornell  Memoir  No.  18. 


RELATION  OF  MICROORGANISMS  TO  SPECIAL  DAIRY  PRODUCTS      515 

If  the  cream  from  which  the  ice  cream  is  made  has  been  produced 
and  handled  under  sanitary  conditions,  the  bacterial  content  should 
consist  chiefly  of  organisms  of  the  Bact.  lactis  acidi  type,  in  which  case 
the  high  count  in  the  ice  cream  might  not  be  objectionable.  If,  on 
the  other  hand,  the  cream  has  been  held  in  cold  storage  for  some  time 
under  conditions  which  inhibit  the  growth  of  the  lactic  organisms  and 
permit  the  development  of  putrefactive  types,  bacterial  poisons  may 
be  developed  in  the  cream,  which  will  be  highly  objectionable.  There 
seems  to  be  little  doubt  that  this  is  the  cause  of  the  cases  of  ptomain 
poisoning,  resulting  from  the  use  of  ice  cream.  It  is  known  that  certain 
types  of  bacteria,  especially  those  belonging  to  the  so-called  putre- 
factive group,  are  capable  of  developing  at  very  low  temperatures  and 
may,  therefore,  produce  considerable  quantities  of  toxic  products  in 
the  cream.  Whether  or  not  these  products  are  developed  before 
the  cream  is  manufactured  or  whether  they  may  develop  in  the  frozen 
product  cannot  at  present  be  stated.  In  general  it  can  be  said  that  the 
total  bacterial  count  does  not  indicate  the  wholesomeness  of  the  ice 
cream  any  more  than  does  a  similar  count  in  buttermilk  or  in  the  com- 
mercial fermented-milk  drinks.  The  kinds  of  organisms  present  is  a 
far  more  important  question  from  the  standpoint  of  the  wholesomeness 
of  the  ice  cream.  However,  the  results  obtained  by  many  ice-cream 
manufacturers  have  demonstrated  the  fact  that  the  germ  content  of  this 
product  can  be  quite  definitely  controlled  by  the  same  methods  of  care 
and  sanitation  as  are  required  in  the  handling  of  other  forms  of  dairy 
products. 


DIVISION  V 
MICROBIOLOGY  OF  FOODS 


CHAPTER  I* 

DESICCATION,  EVAPORATION,  AND  DRYING  OF  FOODS 
AGENCIES  THAT  BRING  ABOUT  CHANGES  IN  DRIED  FOODS 

Food  materials  are  derived  from  plant  and  animal  tissues.  The 
agencies  which  may  bring  about  their  deterioration  are  those 
initially  present  in  the  raw  food,  and  those  introduced  later  in  the 
process  of  handling.  These  agencies  are  the  enzymes  produced  by  the 
cells  constituting  the  food  material,  and  the  bacteria,  yeasts,  and  molds, 
with  their  enzymes,  which  may  be  introduced  later. 

Enzymes  are  normally  present  in  foodstuffs  which  have  not  been 
subjected  to  heating,  since  all  living  cells  apparently  contain  enzymes 
which  may  remain  active  for  a  considerable  length  of  time  after  the 
death  of  the  cells.  These  enzymes  are  usually  termed  autolytic,  that 
is,  they  digest  the  cells  or  parts  of  cells  which  produce  them.  They 
are  active  in  bringing  about  deterioration  of  certain  types  of  foods. 
These  autolytic  enzymes  are  of  many  kinds.  Some  of  them  attack 
carbohydrates,  some  fats,  others  proteins,  and  still  others  organic 
compounds  belonging  to  none  of  these  groups.  They  are,  for  example, 
responsible  for  the  stiffening  of  muscles  after  death  (rigor  mortis], 
and  later  break  down  the  tissues  of  the  meat  and  bring  about  a  so-called 
ripening  whereby  it  becomes  more  tender.  They  may  in  some  instances 
produce  rancidity  in  food  products  by  splitting  of  the  fat.  It  is  self- 
evident,  therefore,  that  if  food  is  to  be  preserved  by  drying,  the  enzymes 
capable  of  bringing  about  detrimental  changes  must  either  be  inhibited 
in  their  action  or  be  destroyed  by  heat  or  by  some  other  agency. 
In  most  cases  the  drying  of  a  food  will  remove  water  sufficiently  to 
inhibit  the  action  of  the 'enzymes  as  well  as  to  prevent  the  growth  of 
microorganisms.  If  the  process  of  desiccation  is  not  properly  carried 

*  Prepared  by  R.  E.  Buchanan. 


DESICCATION,    EVAPORATION   AND   DRYING    OF   FOODS        517 

V 

out  many  color  changes  may  occur  which  interfere  with  the  appearance 
of  the  food  product.  For  example,  it  is  a  common  observation  that  the 
cut  surface  of  an  apple  turns  dark  upon  exposure  to  the  air.  This  is 
due  to  the  presence  of  an  oxidizing  enzyme.  If  a  product  resulting 
from  the  drying  of  apples  is  not  to  be  too  dark  colored,  either  unusual 
care  must  be  used  in  the  preparation  or  some  method  of  inhibiting  the 
action  of  the  enzyme  or  bleaching  the  product  must  be  used.  The 
ripening  process  of  fruits  is  due  to  the  transformation  of  cell  contents  and 
constituents  by  the  enzymes  contained.  When  this  process  continues 
too  long  over-ripening  and  spoilage  occur.  In  other  words,  substances 
which  are  ill-flavored  may  develop  in  foods  which  are  not  dried  rapidly 
enough  or  are  dried  insufficiently. 

Bacteria  are  present  in  large  numbers  upon  the  surfaces  of  many 
raw.  foods.  More  are  added  during  the  process  of  handling.  They 
probably  constitute  the  most  important  single  item  bringing  about 
destruction  or  deterioration,  and  whenever  moisture  and  temperature 
conditions  are  favorable  they  rapidly  bring  about  undesirable  changes. 
Carbohydrates  present  in  the  foods  are  hydrolyzed  and  fermented,  fats 
are  frequently  hydrolyzed,  and  proteins  broken  down  into  simpler 
compounds.  In  general  they  require  somewhat  more  moisture  for 
their  development  than  the  yeasts  and  the  molds. 

Food  materials  may  be  divided  into  two  principal  groups:  those  in 
which  the  desirable  food  constituent  is  in  solution  in  water;  and  those 
in  which  the  principal  food  constituent  is  more  or  less  insoluble.  There 
are  many  foods  which  combine  both  characteristics.  Raisins,  for 
example,  contain  a  considerable  amount  of  sugar  in  solution  in  the 
water  present.  Those  foods  which  are  relatively  insoluble  may  again  be 
divided  into  four  groups,  using  the  amount  of  water  as  a  basis  for 
classification.  First,  those  in  which  moisture  is  present  in  appreci- 
able quantities  in  the  interstices,  that  is,  those  which  seem  wet.  These 
usually  furnish  optimum  conditions  for  the  growth  of  the  bacteria 
which  multiply  rapidly  and  spread  through  the  medium  by  actual 
space  growth,  by  convection  currents,  and  by  their  own  power  of  motion. 
Second,  some  foods  may  contain  moisture  sufficient  for  the  abundant 
growth  of  bacteria  but  not  free  water  which  will  allow  of  rapid  dis- 
tribution. In  these  the  spread  of  the  microorganisms  must  be  largely 
by  direct  growth  and  will  necessarily  be  slower  than  in  the  preceding. 
Third,  the  substance  may  be  so  dry  that  little  or  no  growth  of  the 


518  MICROBIOLOGY  -OF   FOODS 

organisms  may  take  place  yet  there  is  sufficient  moisture  so  that  they 
remain  viable  for  long  periods  of  time.  Fourth,  the  food  may  be  so 
dry  that  only  those  organisms  which  withstand  relatively  complete 
desiccation  will  survive.  These  groups  cannot  be  differentiated  from 
each  other  wholly  on  the  basis  of  the  percentage  of  water  present.  The 
manner  in  which  the  water  is  held,  and  the  substances  which  may  be 
in  solution  in  the  water  are  also  very  important. 

Yeasts  are  present  upon  the  surfaces  of  many  fruits.  They  usually 
require  sugars  for  their  best  development,  and  are  therefore  commonly 
present  in  foods  containing  this  substance.  Yeasts  will  also  be  found 
upon  the  cut  ends  of  twigs  or  grass  culms  where  the  sugary  sap  has  oozed 
out.  Colonies  of  considerable  size  may  sometimes  be  seen  on  corn 
stubble  during  damp  weather.  These  yeasts  are  commonly  distributed 
by  flies  and  other  insects  which  feed  upon  the  plant  juices.  The  yeasts 
are  not  motile,  hence  their  spread  in  any  food  must  be  a?  a  result  of 
direct  growth  or  of  convection  currents. 

Molds,  like  the  bacteria,  are  ubiquitous  and  under  proper  conditions 
will  destroy  most  types  of  food.  They  grow  readily  on  solutions  and 
on  saturated  substrata,  but  frequently  are  overgrown  by  bacteria  under 
these  conditions.  For  example,  wet  silage  rots  when  exposed  to  air 
and  supports  luxuriant  growth  of  bacteria,  while  drier  silage  becomes 
moldy.  Unlike  bacteria  the  molds  extend  through  and  over  food  when 
there  is  no  visible  water  film.  The  spores  are  much  better  adapted  to 
air  dispersal  than  are  bacterial  cells,  and  the  hyphae  penetrate  more 
rapidly  than  will  the  bacterial  colonies.  In  certain  foods,  therefore, 
such  as  meals  and  flours,  molds  are  more  destructive  than  are  bacteria. 
Usually  they  will  multiply  with  somewhat  less  moisture. 

FACTORS  WHICH  INHIBIT  GROWTH  OF  MICROORGANISMS  IN  DESICCATED 

FOODS 

The  factors  which  appear  to  be  of  greatest  importance  in  inhibiting 
the  growth  of  microorganisms  in  dried  foods  are:  the  relatively  complete 
absence  of  free  water,  concentration  of  solutes,  formation  of  water- 
free  protective  layers,  and  the  action  of  heats,  sunlight,  sulphur  dioxide, 
smoke,  or  other  disinfectants  or  bleaching  agents. 

The  amount  of  water  remaining  in  a  desiccated  food  is  probably  the 
most  important  single  factor  in  determining  its  keeping  qualities.  In  a 


EVAPORATION   AND   DRYING    OF   FOODS        519 

few  cases  the  development  of  microorganisms  is  absolutely  inhibited 
by  the  absence  of  sufficient  moisture  in  the  food  to  support  growth. 
Many  foods  which  appear  to  be  dry  nevertheless  contain  an  appreciable 
amount  of  moisture.  The  amount  necessary  to  bring  about  appreci- 
able changes  or  detrimental  changes  is  sometimes  not  very  great.  For 
example,  the  amount  of  moisture  present  in  raw  sugar  appears  to  con- 
stitute a  real  loss  to  the  manufacturer.  Some  foods,  such  as  olive  oil, 
starches,  meals,  cane  sugar,  etc.,  have  little  or  no  free  water.  Others 
contain  an  appreciable  amount  of  water  and  yet  do  not  deteriorate, 
usually  because  the  drying  has  resulted  in  a  concentration  of  the  solutes 
beyond  the  point  to  which  the  microorganisms  can  adapt  themselves 
to  the  osmotic  pressure.  When  it  is  remembered  that  a  50  per  cent 
solution  of  cane  sugar  is  capable  of  exerting  a  pressure  of  about  225 
kilograms  per  square  inch,  it  will  be  realized  that  considerable  capa- 
city for  readjustment  is  necessary  in  the  cell  of  any  yeast  or  mold  that 
can  grow  in  such  a  medium. 

In  the  process  of  drying,  the  former  relationships  of  tissue  cells 
and  tissue  constituents  may  be  so  changed  that  protective  layers  are 
formed.  For  example,  in  curing  pork,  the  fat  which  was  structurally 
isolated  in  distinct  cells  for  the  most  part  becomes  diffused  through 
the  outer  layers  of  the  tissues  and  forms  a  water-free  and  water-proof 
exterior.  The  keeping  quality  of  the  dried  food  is  sometimes  in  part 
dependent  on  the  destruction  of  microorganisms  by  heat  during  the 
process  of  drying.  In  other  cases  they  are  exposed  to  the  germicidal 
action  of  the  direct  rays  of  the  sun,  or  to  the  action  of  some  disinfectant 
or  bleaching  agent,  such  as  sulphur  dioxide  or  smoke. 

METHODS  OF  DRYING 

The  rapidity  with  which  foods  may  be  dried  depends  upon  the 
amount  of  water  present  in  the  food,  the  texture  and  size  of  the  particles, 
the  temperature,  the  relative  humidity  of  the  atmosphere,  and  the 
rapidity  of  the  current  of  air  which  carries  away  the  moisture.  The 
rapidity  with  which  foods  must  be  dried  in  order  to  remain  palatable 
depends  in  part  upon  how  subject  they  are  to  the  attacks  of  molds, 
yeasts,  or  bacteria,  in  part  upon  the  rapidity  of  ghanges  brought  about 
by  autolytic  enzymes,  and  in  part  upon  the  changes  in  flavor  and  tex- 
ture which  may  be  brought  about  by  the  application  of  too  high 
a  temperature. 


520  MICROBIOLOGY    OF    FOODS 

A  reduction  of  the  water  in  foods  below  the  minimum  required  for 
the  growth  of  microorganisms  is  accomplished  in  a  variety  of  ways. 
Most  commonly  heat  is  employed,  either  the  sun's  rays  or  from  some 
artificial  source.  In  localities  where  the  humidity  of  the  air  is  low,  as  in 
many  of  the  irrigated  fruit  districts  of  the  western  United  States, 
exposure  to  the  rays  of  the  sun  is  sufficient  for  drying.  For  other  types 
of  foods,  and  in  more  humid  regions,  artificial  heat  is  used  to  reduce  the 
relative  humidity.  Some  foods  cannot  be  dried  at  high  temperatures 
because  of  their  instability.  They  are  usually  dried  at  a  low  tempera- 
ture and  in  a  partial  vacuum.  Other  foods  are  dried  without  recourse 
to  evaporation  by  the  use  of  hydraulic  presses  or  by  centrifugation,  the 
latter  in  the  manufacture  of  cane  sugar.  The  water  for  the  growth  of 
microorganisms  may  be  reduced  by  the  addition  of  some  crystalline 
substance  such  as  sugar  or  salt.  The  usefulness  of  the  latter  method 
depends  largely  upon  the  creation  of  a  concentration  of  solutes  too 
great  for  the  growth  of  the  bacteria.  At  the  same  time  a  considerable 
proportion  of  the  water  from  that  part  of  the  food  into  which  the 
solutes  will  not  penetrate  is  extracted  by  osmosis. 

Many  food  products  do  not  require  any  special  drying  as  they 
naturally  contain  little  moisture.  Such  are  the  grains  and  the  products 
manufactured  from  them,  as  flour.  The  drying  in  this  instance  has 
occurred  during  and  immediately  following  the  ripening  process  of  the 
grain.  When  for  any  reason  this  does  not  occur  the  grain  will  mold. 
It  has  been  found  necessary  in  many  instances  to  kiln-dry  corn. 
Grains,  nuts,  etc.,  are  by  their  nature  adapted  to  keep  under  normal 
conditions  for  considerable  periods,  although  there  is  usually  present  in 
nuts  sufficient  moisture  to  allow  of  the  slow  action  of  lipolytic  enzymes 
and  consequent  development  of  rancidity.  Other  foods  require  artifi- 
cial drying.  In  these  we  have  the  intergrading  classes  which  we  have 
discussed  above;  those  which  contain  a  small  percent  of  water  and  those 
which  contain  considerable  water  but  a  high  concentration  of  solutes. 
The  absolute  amount  of  water  in  a  food  is  by  no  means  the  index  to  the 
amount  that  is  available  for  the  growth  of  microorganisms.  Many 
foods  are  hygroscopic.  Foods  having  the  same  water  content  and 
percentage  of  solute^  will  behave  very  differently  with  reference  to 
delivering  up  the  water  to  an  organism  present. 

The  effect  of  the  concentration  of  solutes  by  drying  is  perhaps 
the  most  important  factor  in  the  preservation  of  foods.  These  sub- 


DESICCATION,    EVAPORATION   AND    DRYING    OF   FOODS        521 

stances  dissolved  in  the  water  may  be  actually  antiseptic  when  concen- 
trated, as  the  acids  of  the  juices  of  certain  fruits.  More  often  the 
sugars  reach  a  concentration  so  great  as  to  prevent  growth  by  plasmo- 
lyzing  the  cell  contents  of  the  organism.  For  every  organism  there  is  a 
maximum  concentration  reached  sooner  or  later  beyond  which  growth 
is  impossible. 

i  Dried  foods  may  be  divided  into  three  groups  using  the  relative 
abundance  of  carbohydrates,  fats,  and  proteins,  as  the  basis 
of  classification. 

Carbohydrate  foods  are  usually  preserved  by  drying.  Many,  such  as 
grains  and  nuts  and  the  flours  and  meals  prepared  from  them,  do  not  re- 
quire artificial  heating.  They  are,  however,  somewhat  hygroscopic  and 
in  damp  climates  enough  moisture  is  taken  up  to  allow  the  growth  of 
injurious  molds  and  bacteria.  Still  other  carbohydrate  food  stuffs 
require  more  or  less  care  in  the  drying  or  curing,  such  as  hay  and  fodder 
in  general.  These  are  usually  dried  by  exposure  to  the  air  and  sun 
until  most  of  the  water  has  been  evaporated.  Fodder  that  has  become 
moldy  through  the  presence  of  too  much  moisture  is  the  cause  of  trouble 
in  horses  and  less  frequently  in  cattle.  Many  deaths  due  to  the  so- 
called  cerebro-spinal  meningitis  in  the  horse  are  frequently  due  to  the 
consumption  of  moldy  hay.  In  localities  where  the  air  is  too  moist  or 
rains  so  frequent  as  to  make  it  difficult  to  dry  hay,  curing  is  effected 
by  a  process  of  self -fermentation.  The  hay  is  piled  in  a  mass  while 
still  green  and  undergoes  a  process  of  heating.  The  temperature  rises  to 
about  70°.  The  cause  of  this  rise  is  somewhat  uncertain  but  is  probably 
due  to  the  combined  action  of  enzymes  and  microorganisms.  Just  how 
much  of  the  keeping  quality  is  due  to  the  heating,  how  much  to  the  loss 
of  water,  and  how  much  to  the  accumulation  of  products  of  fermenta- 
tion is  uncertain.  In  other  cases  the  heated  hay  is  spread  out  and 
quickly  dried  sufficiently  so  that  it  may  be  stored.  A  certain  small 
percentage  of  the  nutriment  in  the  hay  is  necessarily  lost  in  the  develop- 
ment of  the  heat. 

Many  vegetables  in  desiccated  form  can  now  be  bought  upon  the 
market,  and  have  been  prepared  in  recent  years  in  large  quantities  for 
household  use  by  the  housewife.  Fruits  are  also  quite  generally  pre- 
served by  drying.  In  many  instances,  as  in  peaches,  apples,  and  berries, 
it  is  probable  that  enough  moisture  is  removed  to  prevent  organisms 
from  growing,  but  in  many  other  cases,  as  in  the  preparation  of  currants 


522  MICROBIOLOGY   OF   FOODS 

and  raisins,  the  concentration  of  sugar  and  other  solutes  is  the  control- 
ling factor.  Frequently  as  much  as  30  per  cent  of  the  dried  fruit  is 
water.  Fruit  drying  is  often  accomplished  by  heating  in  the  sun's 
rays,  in  other  cases  artificial  heat  and  even  hydraulic  pressure  are 
used. 

Many  manufactured  products,  particularly  bakers'  goods  such  as 
crackers,  biscuits,  dried  yeast  cakes,  etc.,  are  preserved  by  the  elimina- 
tion of  water. 

Macaroni  and  vermicelli  are  prepared  by  forcing  a  thick  paste  of  especially  pre- 
pared flour  and  water  through  openings  of  different  sizes.  The  product  is  then 
dried  in  the  air  until  it  is  brittle  and  may  then  be  kept  indefinitely. 

Copra,  one  of  the  principal  exports  of  certain  of  the  islands  of  the  Pacific  and 
Indian  oceans,  is  prepared  by  cutting  the  meat  of  the  cocoanut  into  pieces  and  dry- 
ing in  the  sun.  From  this  copra  much  of  our  desiccated  and  powdered  cocoanut  is 
prepared,  and  from  it  is  pressed  the  cocoanut  oil  which  finds  so  many  uses  in  manu- 
facture. 

Syrups,  molasses,  jellies,  jams,  and  many  other  carbohydrate  foods 
are  preserved  through  the  concentration  of  solutes.  Many  of  these  are 
partially  sterilized  by  the  heat  used  in  the  process  of  manufacturing. 
There  is  usually  plenty  of  opportunity  for  subsequent  infection.  They 
are  more  frequently  attacked  by  molds  and  yeasts  than  bacteria.  An 
exception  may  be  noted  in  Leuconostoc  mesenterioides,  a  bacterium  which 
causes  considerable  trouble  by  a  gelatinous  fermentation  in  syrups  from 
which  sugars  are  being  manufactured. 

Fatty  foods  frequently  contain  little  water.  Cottonseed,  olive, 
cocoanut,  and  other  vegetable  oils,  the  plant  and  animal  fats  as  lard, 
tallow  and  butter,  are  quite  resistant  to  change  by  bacteria  unless 
water  is  present  and  considerable  traces  of  nitrogenous  materials 
remain  in  them.  With  these  foods  the  water  is  necessary  for  the 
growth  of  the  organism  and  also  for  the  action  of  the  lipoly tic  enzymes 
which  might  hydrolyze  fats  and  aid  in  the  development  of  rancidity. 
Butter  is  an  exception  to  the  rule  that  fatty  foods  contain  little  water,  as 
it  usually  has  from  12  per  cent  to  16  per  cent.  When  it  is  necessary 
to  keep  butter  fat  for  long  periods  under  unfavorable  conditions, 
the  water  and  nitrogenous  material  are  removed  and  the  clear  fat 
preserved.  This  is  the  so-called  ghee  of  India.  Bacteria,  enzymes,  and 
a  few  molds  have  been  described  that  attack  fat.  In  the  process  of 
preparation  or  manufacture  of  any  fat  foods  sufficient  heat  may  be  used 
to  .sterilize  the  material,  and  infection  thereafter  penetrates  to  the 
interior  very  slowly.  This  heat  destroys  the  enzymes  as  well  as  the 
bacteria. 


DESICCATION,    EVAPORATION   AND   DRYING    OF   FOODS        523 


Protein  foods  are  in  large  part  flesh  foods  and  flesh  derivatives. 
Desiccation,  however,  is  only  one  of  the  agencies  acting  to  preserve 
the  flesh. 

Jerked  meat  is  sometimes  prepared  in  localities  with  a  hot  dry  climate.  Lean 
meat  is  cut  into  thin  slices  and  exposed  to  the  direct  rays  of  the  sun  until  dry.  The 
bactericidal  action  of  the  sunlight  and  the  rapid  extraction  of  moisture  prevents 
microorganisms  from  producing  undesirable  changes  during  the  curing  process. 

Dried  beef  is  lean  meat  which  usually  has  been  treated  with  certain  condiments  or 
smoked  and  salted  and  then  dried. 

Dried  fish  such  as  cod,  mackerel,  and  herring,  is  prepared  by  the  use  of  condi- 
ments, salt,  and  smoke  in  addition  to  the  drying. 

Pemmican  is  prepared  by  drying  lean  meat,  grinding  it,  and  mixing  it  with  sugar 
and  fat,  dried  fruits,  spices,  etc.  It  is  highly  nutritious,  not  unpalatable,  and  com- 
pact, and  will  keep  for  a  long  period.  It  is  frequently  used  as  a  concentrated  form 
of  food  by  Arctic  explorers,  etc. 

Beef  extract  is  prepared  by  cooking  minced  beef  and  water  in  a  receptacle  under 
a  slight  steam  pressure.  The  digestion  is  continued  for  several  hours.  The  liquid  is 
filtered  off  and  concentrated  in  a  partial  vacuum  to  the  desired  consistency. 

Gelatin  is  prepared  by  boiling  bones  and  tendons,  sometimes  also  horn  and  hide 
scraps  and  concentrating  the  gelatin  which  dissolves  from  these. 

Somatose,  sarco-peptone  and  related  so-called  predigested  protein  foods  are  mix- 
tures of  albumoses  and  peptones  prepared  by  the  artificial  digestion  and  drying  of 
proteins,  usually  flesh.  The  product  is  marketed  as  a  powder. 

Milk,  either  with  or  without  its  butter  fat,  is  dried  by  being  sprayed  into  a  warm 
compartment  from  which  the  air  is  partly  exhausted.  It  dries  immediately,  in  the 
form  of  a  very  fine  powder.  This  powder,  if  thoroughly  dry,  will  keep  well  and  is 
finding  an  extensive  use.  The  high  sugar  content  of  this  powder  is  instrumental  in 
preventing  the  development  of  microorganisms. 

Eggs  are  dried  in  much  the  same  manner  as  milk  and  the  product  is  being  used 
extensively  at  the  present  time  by  bakers. 

Meats  are  frequently  preserved  by  a  combination  of  drying  and  the 
action  of  certain  antiseptics  or  preservatives.  The  salting  of  meat  owes 
its  effectiveness  in  part  to  the  abstraction  of  water.  In  most  cases, 
the  surface  of  the  meat  and  probably  even  the  other  portions  are 
protected  in  large  measure  by  the  diffusion  of  the  fat  and  the  satura- 
tion of  tissues  and  by  the  formation  of  water-proof  fat  films.  The 
autolytic  enzymes  are  active  in  the  fresh  meat  and  soon  become 
inert  upon  the  removal  of  water.  The  organisms  responsible  for 
decay  of  preserved  meats  and  flesh  foods  are  usually  bacteria.  Some 
of  these  break  down  the  protein  into  simpler  chemical  compounds,  of 
which  a  few  are  known  to  be  poisonous. 


CHAPTER  II* 
HEAT  IN  THE  PRESERVATION  OF  FOOD  PRODUCTS 

HISTORICAL  RESUME 

The  principle  involved  in  the  preservation  of  food  by  heat  may  be 
said  to  have  had  its  origin  in  the  experiments  of  Spallanzani,  who  in 
1765  boiled  meat  extract  for  an  hour  and  hermetically  sealed  the  flasks, 
after  which  treatment  no  change  occurred  in  the  material.  An  applica- 
tion of  this  principle  was  suggested  as  early  as  1782  by  the  Swedish 
chemist,  Scheele,  who  advised  the  exposure  of  vinegar  in  bottles  to  the 
temperature  of  boiling  water  in  order  to  effect  its  preservation.  Some 
years  later  the  principle  was  applied  to  the  conservation  of  food  by  a 
French  confectioner,  Nicholas  Appert,  who  in  1811  published  an 
exhaustive  treatise  on  "The  Art  of  Preserving  Animal  and  Vegetable 
Substances."  His  method  was  to  enclose  the  food  in  a  glass  jar  which 
was  then  corked  tightly,  and  placed  in  boiling  water,  the  length  of 
time  of  heating  varying  with  the  article  to  be  treated. 

In  1810  Peter  Durand  secured  a  patent  from  the  English  govern- 
ment for  the  preservation  of  fruits,  vegetables  and  fish  in  hermetically 
sealed  tin  and  glass  cans.  He  did  not  claim  to  be  the  discoverer  of  the 
process,  but  said  it  had  been  communicated  to  him  by  a  "  foreigner 
residing  abroad."  Although  the  secret  of  the  process  was  jealously 
guarded,  the  employees  of  different  establishments  became  familiar 
with  its  essentials,  and  in  this  manner  the  industry  found  its  way  to 
America.  One  of  the  first  to  introduce  the  process  was  Ezra  Daggett, 
who,  with  his  son-in-law  Thomas  Kensett,  in  1819  engaged  in  the  manu- 
facture of  hermetically  sealed  goods,  the  principal  foods  packed  being 
salmon,  lobsters,  and  oysters.  In  1820,  William  Underwood  and 
Charles  Mitchell,  emigrant  employees  from  a  canning  factory  in  England, 
opened  a  factory  in  Boston  where  they  canned  plums,  quinces,  cran- 
berries and  currants. 

*  Prepared  by  S.  F.  Edwards. 

524 


HEAT   IN   THE    PRESERVATION    OF   FOOD    PRODUCTS  525 

In  the  earliest  days  of  canning,  glass  jars  were  used  exclusively,  but 
were  gradually  abandoned  as  it  was  found  that  they  could  not  readily 
withstand  the  extremes  of  temperature,  and  were  expensive,  bulky, 
and  costly  in  transportation.  In  1825,  Thomas  Kensett  secured  a 
patent  on  the  use  of  tin  cans  in  preserving  food,  and  in  the  same  year 
began  using  the  process  in  his  factory.  The  early  manufacture  of  tin 
cans  was  by  hand  and  crude,  the  bodies  being  cut  with  shears  and  the 
side  seams  made  with  a  plumb  joint  (that  is  meeting  but  not  over- 
lapping), and  then  soldered  together.  Heads  were  made  to  set  into 
the  body  and  were  soldered  in  place  in  a  very  crude  manner.  The 
making  of  100  cans  was  considered  a  good  day's  work  for  one  man. 
Improvements  were  gradually  made,  however,  in  their  manufacture, 
until  finally  can  making  became  a  distinct  industry  and  now  all  the 
parts  are  made  and  put  together  by  machinery. 

In  the  original  Appert  process,  the  goods  were  cooked  in  open  kettles, 
the  highest  temperature  obtainable  by  this  method  being  the  boiling 
point.  A  little  later  common  salt  was  used  to  aid  in  securing  a  higher 
temperature,  and  this  was  followed  later  by  the  use  of  calcium  chloride 
which  made  possible  a  temperature  of  115°.  In  1874,  a  closed  kettle 
was  invented  for  superheating  water  with  steam,  and  this  was  imme- 
diately followed  by  another  improved  kettle  in  which  dry  steam  was 
used,  the  principle  employed  being  that  of  the  modern  autoclav,  by 
which  method  any  desired  temperature  may  be  obtained  and  modified 
to  suit  the  requirements  of  different  classes  of  food. 

ECONOMIC  IMPORTANCE 

FROM  STANDPOINT  OF  HEALTH  AND  DIETETICS. — The  value  of  a 
variety  of  foods,  especially  fruits  and  vegetables,  is  recognized  by 
dietitians.  Unfortunately,  however,  the  season  for  fresh  fruits  and 
vegetables  is  comparatively  short.  Moreover,  many  foods  grown 
exclusively  in  one  section  of  country  will  not  withstand  shipping  in  a 
fresh  condition  to  other  sections.  In  spite  of  improved  methods  of 
refrigeration,  it  is  n6t  practicable  to  ship  fresh  sea  foods  to  far  inland 
towns,  or  to  send  some  perishable  products  of  warm  climates  to  cold 
countries.  The  canning  and  preserving  industry  overcomes  these 
difficulties  by  supplying  pure,  clean,  wholesome  fruits,  vegetables, 
meats,  and  fish  to  any  region  the  year  round,  and  at  prices  com- 
paratively low. 


526  MICROBIOLOGY   OF   FOODS 

FROM  STANDPOINT  OF  COMMERCE. — In  its  commercial  aspect,  the 
importance  of  the  industry  can  scarcely  be  estimated.  Canned 
products  make  possible  the  carrying  of  larger  stores  of  provisions  by 
armies  and  navies  and  expeditions  for  exploration  than  would  otherwise 
be  possible.  In  fact,  the  stimulus  which  prompted  the  investigation  of 
Appert  was  a  prize  offered  by  the  French  Navy  Department  for  a 
method  of  preserving  foods  for  provisioning  ships  more  satisfactory 
than  pickling,  drying,  smoking  or  preserving  in  sugar,  the  methods 
in  use  up  to  that  time. 

"Although  the  preserving  industry  was  established  in  three  great 
commercial  centers  in  the  United  States  as  early  as  1825,  it  did  not 
become  of  much  importance  until  the  last  decades  of  the  nineteenth 
century.  There  were  many  hindrances  to  the  progress  of  the  industry, 
such  as  the  secrecy  observed  in  the  process,  skepticism  of  the  public 
regarding  the  healthfulness  of  canned  foods,  the  general  prejudice 
against  them,  and  the  high  cost  of  production.  These  obstacles  have 
gradually  been  surmounted,  and  at  the  present  time  the  several 
branches  of  the  industry  have  collectively  assumed  large  proportions. 

An  idea  of  the  magnitude  and  importance  of  the  industry  in  the 
United  States  may  be  gained  from  statistics  for  1918  compiled  by  the 
National  Canners'  Association,  and  here  reproduced  by  permission. 
The  pack  of  tomatoes  was  15,882,372  cases;  of  corn,  11,721,860  cases; 
and  of  peas,  10,898,222  cases.  The  total  vegetable  pack  for  1917  other 
than  corn,  peas,  and  tomatoes,  was  13,391,294  cases,  and  of  fruit  was 
11,285,659  cases.  The  average  case  holds  two  dozen  cans.  These 
figures  do  not  include  the  pack  of  oysters,  meats,  or  fish.  The  total 
annual  consumption  of  canned  foods  has  been  estimated  at  250,000,000 
cases.  It  is  apparent  from  these  figures  that  the  canning  and  preserv- 
ing, industry  is  one  of  immense  value,  and  that  it  constitutes  a  large 
factor  in  the  feeding  of  the  world. 

ALTERATION  OF  FOOD 

PHYSICAL  CHANGES. — Appearance.— Some,  physical  changes  attend 
the  conservation  of  foods  by  heat,  approaching  more  or  less  closely  the 
changes  incident  to  the  ordinary  preparation  of  fresh  foods  for  the 
table.  In  the  preserving  of  some  fruits  and  vegetables  the  canner 
subjects  them  to  a  blanching  or  fore-cooking  process  which  consists  in 
submitting  the  product  to  the  action  of  hot  water  for  a  short  time.  The 


HEAT    IN    THE    PRESERVATION    OF   FOOD    PRODUCTS  527 

object  of  blanching  is  first,  for  the  purpose  of  removing  the  more  or  less 
gummy  substance  upon  the  surface  of  such  vegetables  as  peas  and 
beans;  second,  to  make  the  product  more  or  less  flexible  so  that  it  may 
be  packed  without  breaking,  as  asparagus;  third,  to  permit  the  packing 
of  a  greater  quantity  in  a  can,  as  spinach;  fourth,  to  force  water  into 
the  product  and  cause  it  to  be  tender,  as  in  beans;  fifth,  to  secure  a 
more  uniform  color,  as  in  fruits;  and  sixth,  for  its  cleansing  effect.  It 
is  not  a  bleaching  process  as  many  infer  from  the  term.  The  time  used 
in  blanching  varies  from  one  to  fifty  minutes,  usually  being  between 
two  and  five  minutes.  The  operation  is  of  no  value  in  reducing  the 
time  necessary  to  properly  process  canned  foods. 

Mechanical  Disintegration. — In  the  case  of  very  soft  fruits  or  vege- 
tables, the  high  temperature  of  processing  causes  a  slight  amount  of 
mechanical  disintegration,  which  is  not  objectionable  unless  exces- 
sive, as  there  is  little  deterioration  in  appearance  and  none  at  all  in 
food  value.  In  the  case  of  meats,  practically  the  only  physical  change 
is  the  shrinkage  during  the  parboiling  previous  to  placing  in  the  cans. 

CHEMICAL  CHANGES.  Appearance. — The  chemical  changes  in  foods 
preserved  by  heat  may  be  considered  under  two  heads:  first,  those  in 
which  the  appearance  is  modified;  and  second,  those  in  which  the  food 
itself  is  altered.  Some  change  of  color  sometimes  occurs  and  results 
from  various  causes.  In  colored  vegetables,  such  as  peas,  string  beans, 
and  asparagus,  a  part  at  least  of  the  loss  of  color  is  due  to  the  oxidation 
of  chlorophyll.  With  a  few  foods,  iron  sulphides  are  occasionally 
formed  by  a  combination  of  sulphur  with  the  iron  of  the  container. 
This  seldom  occurs,  however,  and  is  not  of  great  importance.  Some 
fruits  packed  in  glass  gradually  lose  their  color  by  oxidation  on  exposure 
to  the  light. 

Chemical  Change. — So  far  as  chemical  alteration  of  the  food  itself 
is  concerned,  there  is  little  change  and  none  other  than  would  occur  in 
the  preparation  of  the  food  for  the  table.  The  albumins  are  coagulated. 
The  fats  probably  remain  unchanged.  Of  the  carbohydrates,  the  chief 
action  is  on  the  sugars.  The  cane  sugar  is  wholly  or  partly  inverted 
by  the  combined  action  of  the  heat  and  the  fruit  or  vegetable  acids. 
The  starch  undergoes  little  if  any  cleavage,  inasmuch  as  this  change 
occurs  only  in  the  presence  of  acids  and  in  foods  with  a  relatively 
high  acid  content,  the  proportion  of  starch  is  low.  The  other  amyloses 
undergo  little  if  any  change. 


528  MICROBIOLOGY    OF    FOODS 

Palatability  and  Digestibility. — It  is  often  contended  that  canned 
foods  are  less  palatable  than  fresh  foods  of  the  same  kind.  This  lack 
of  agreeableness  to  the  taste  is,  however,  more  seeming  than  real,  and 
arises  largely  from  the  prejudice  of  the  consumer  against  food  conserved 
in  tin  cans  rather  than  from  any  actual  change.  When  the  preserving 
is  properly  done  the  product  should  be  no  less  attractive  to  the  eye,  no 
less  pleasing  to  the  palate,  and  of  no  less  value  from  the  standpoint  of 
digestibility  than  the  same  food  when  served  in  the  fresh  condition. 

BIOLOGICAL  CHANGES.  Vital  Disorganization. — The  entire  industry 
of  conservation  of  food  by  means  of  heat  is  based  on  a  microbiological 
process.  It  is  a  universally  recognized  fact  that  the  ordinary  spoilage  of 
food  is  a  microbiological  change,  hence  to  protect  food  from  spoilage 
consideration  must  be  given  to  the  microbial  agents  responsible  for 
the  change. 

Normal  Flora  and  Fauna. — Unlike  some  branches  of  microbiology 
as  medical  or  dairy,  we  are  unable  to  designate  definite  species  as  those 
usually  identified  with  the  spoilage  of  canned  foods.  Considering 
the  great  variety  of  foods  preserved  by  heat,  and  the  different  con- 
ditions under  which  they  are  grown  and  secured,  it  naturally  follows 
that  the  normal  flora  and  fauna  of  food  to  be  preserved  in  this  manner 
would  embrace  a  wide  variety  of  species,  including  some  higher  fungi, 
molds,  yeasts,  bacteria,  and  low  animal  forms.  Generally  speaking, 
the  microbial  flora  of  fruits  consists  mostly  of  molds  and  yeasts, 
although  bacterial  forms  may  also  be  present.  In  the  case  of  vege- 
tables, and  of  fruits  coming  in  contact  with  the  earth,  more  species  of 
bacteria  are  apt  to  be  present,  many  of  them  spore  formers  able  to 
withstand  a  high  temperature.  Finally,  in  meats  and  fish  the  living 
forms  may  include  not  only  molds,  yeasts,  and  bacteria,  but  animal 
forms  as  well,  such  as  the  organisms  of  taeniasis  (tapeworm)  and  trichi- 
nosis. Weinzirl,  Hunter  and  Thorn,  Sadler,  and  others,  by  investiga- 
tions reported  in  1918  and  1919  showed  that  the  bacteria  most  often 
present  in  canned  foods  were  of  the  B.  mesentericus  type,  and  those  of 
the  colon  group.  Weinzirl  made  bacteriological  examinations  of  1018 
samples  of  canned  foods  including  spoiled  goods,  experimental  under- 
processed  samples,  and  market  samples.  "The  organisms  isolated 
comprised  (a)  yeasts,  17  cultures,  (b)  molds,  29  cultures  representing 
7  genera,  and  (c)  bacteria,  392  cultures  representing  38  species  of  which 
B.  mesentericus  (Flugge)  was  the  most  prevalent."  Hunter  and  Thorn 


HEAT   IN   THE   PRESERVATION   OF   FOOD   PRODUCTS  529 

examined  530  cans  of  salmon  representing  9  brands,  and  found  237 
unsterile  cans;  224  of  these  contained  the  same  organism  of  the  B. 
mesentericus  group,  either  in  pure  culture  or  in  connection  with  other 
species.  Tinned  sardines  show  a  high  percentage  of  organisms  of  the 
colon  group.  As  the  intestines  are  not  removed  before  the  fish  are 
packed,  this  would  naturally  be  expected.  Weinzirl  concluded  that 
food  poisoning  organisms  such  as  B.  botulinus  and  B.  enteritidis,  etc., 
are  not  found  in  commercial  canned  foods.  Burke,  however,  in  1919, 
examined  235  cultures  from  a  wide  range  of  material  in  California, 
including  tap  water,  hay,  leaves,  vegetables,  fruits  in  various  conditions, 
insects,  spiders,  sowbugs,  snails  and  caterpillars,  garden  soil,  manure 
from  horses,  hogs  and  chickens,  and  also  samples  from  the  claws  and 
beaks  and  from  the  crop,  gizzard  and  intestinal  contents  of  birds. 
Seven  cultures  containing  B.  botulinus  were  found.  Burke  concluded 
from  her  research  that  "B.  botulinus  is  widely  distributed  in  nature; 
that  it  is  present  in  the  garden  and  may  be  on  the  fruits  or  vegetables 
when  they  are  gathered."  Bigelow  and  Estey,  in  a  paper  read  before 
the  Society  of  American  Bacteriologists  in  December,  1919,  emphasize 
the  importance  of  further  knowledge  of  the  thermophilic  organisms. 
"  These  bacteria  are  frequently  mentioned  but  practically  nothing  has 
been  done  with  them.  We  have  quite  a  number  of  them  isolated,  some 
of  them  being  acid  formers  and  producing  "flat  sours,"  and  others 
being  gas  forming.  One  of  them  converts  starch  to  maltose  and  pro- 
duces the  so-called  "sweet  hominy."  Another  one  turns  milk  bitter 
and  has  caused  some  spoilage  in  evaporated  milk  which  was  not 
recognized  as  bacterial  spoilage.  Some  of  these  organisms  do  not  appear 
to  grow  below  the  temperature  of  42°C.,  and  grow  as  high  as  76°C. 
Others  grow  readily  at  65°C.  and  as  low  as  room  temperature.  We  do 
not  know  how  much  higher  or  lower.  These  resistant  spores  do  not 
appear  to  grow  at  a  lower  pH  value  than  about  4.7.  They  therefore 
are  not  expected  to  give  any  difficulty  in  processing  products  as  acid  as 
tomatoes  or  as  the  ordinary  fruits.  The  lesson  from  the  data  secured 
is  that  foods  should  be  processed  at  as  high  a  temperature  as  possible. 
These  resistant  organisms  require  many  hours  for  the  destruction  of 
their  spores.  The  spores  are  not  destroyed  by  fractional  sterilization. 
If  these  spores  are  present  and  a  high  temperature  is  not  used  for  their 
destruction,  they  will  cause  spoilage." 


530  MICROBIOLOGY   OF   FOODS 

PASTEURIZATION 

ECONOMIC  CONSIDERATIONS. — In  the  preservation  of  food  by  heat, 
two  processes  are  applicable,  pasteurization  and  processing  or  steriliz- 
ation. In  pasteurization,  the  aim  is  not  to  effect  the  permanent 
preservation  of  foods  or  drinks  by  destroying  all  life  present,  but 
rather  to  destroy  certain  species  of  organisms,  thus  checking  the 
natural  fermentation,  and  effecting  a  temporary  preservation. 

The  principle  of  pasteurization  may  be  said  to  have  originated  in 
the  early  work  of  Spallanzani  and  Scheele,  already  mentioned,  and 
was  employed  by  Appert  in  his  later  investigations.  The  operation 
as  carried  out  by  Appert  does  not,  however,  appear  to  have  found 
general  application  until  Pasteur  revived  the  method,  and  as  a  result 
of  his  activities  in  attempting  to  secure  a  general  adoption  of  the 
practice  to  prevent  the  spoiling  of  wine,  the  process  was  named  from 
him. 

SPECIFIC  APPLICATION.  Beer. — Pasteurization  is  of  economic  im- 
portance particularly  in  the  dairy  and  fermentation  industries,  and 
has  perhaps  had  its  widest  application  in  the  brewing  industry.  The 
method  as  stated  by  the  Schlitz  Brewing  Company  is  as  follows: 
"  The  process  of  pasteurization  is  in  use  with  even  the  smallest  brewers 
in  the  United  States,  beer  being  pasteurized  even  for  local  consumption. 
The  beer  is  pasteurized  in  bottles  by  being  subjected  to  a  temperature 
of  58°  to  63°  for  one-half  hour.  The  entire  process  as  practised  in  the 
large  breweries  requires  less  than  an  hour,  and  includes  the  warming  of 
the  cold  bottles  to  pasteurizing  temperature,  the  pasteurizing  proper, 
and  the  cooling  down  to  a  little  above  room  temperature.  The  process 
is  a  continuous  one,  the  bottles  being  put  into  the  machine  at  one  end 
and  taken  out  at  the  other." 

Fruit  Juices. — The  essentials  in  the  pasteurization  of  wine  and  fruit 
juices  are  similar  to  those  for  beer.  There  is^  however,  no  universal 
rule  of  application.  Details  of  the  process  must  be  worked  out  to  suit 
the  character  of  the  different  liquids  under  treatment. 

Cream  and  Milk. — Pasteurization  as  employed  in  the  dairy  industry 
varies  in  its  method  of  application  according  to  the  purpose  for  which 
it  is  used.  Milk  or  cream  as  ordinarily  received  at  creameries  contains 
a  widely  variant  microbial  flora,  many  of  the  species  exerting  a  greater 
or  lesser  influence  in  determining  the  flavor  of  the  finished  product. 


HEAT   IN    THE    PRESERVATION    OF    FOOD    PRODUCTS  531 

By  pasteurization  of  the  cream,  the  butter-maker  destroys  most  of  the 
organisms  present;  and  by  the  use  of  a  culture  starter  of  lactic  acid 
bacteria,  he  is  able  to  control  the  fermentation,  and  is  assured  of  a 
uniform  quality  of  product  from  day  to  day  throughout  a  season.  An 
added  value  of  pasteurization  is  that  all  pathogenic  organisms  are 
destroyed,  thus  aiding  in  the  prevention  of  such  diseases  as  might  be 
conveyed  through  this  product.  In  creameries,  the  usual  method  of 
pasteurization  is  what  is  known  as  the  continuous  or  flash  process,  in 
which  the  milk  is  subjected  to  a  momentary  heating  to  about  85°, 
the  flow  of  milk  through  the  pasteurizing  machine  being  so  regulated  as 
to  bring  all  the  milk  up  to  the  desired  temperature,  the  heating  being 
immediately  followed  by  rapid  cooling,  and  subsequent  addition  of  the 
lactic  starter. 

In  the  pasteurization  of  milk  for  infant  feeding,  a  lower  temperature 
is  employed.  A  temperature  sufficiently  high  to  kill  the  organism  of 
tuberculosis  (the  standard  for  pasteurization)  by  momentary  heating, 
imparts  to  the  milk  a  cooked  flavor,  making  it  less  palatable,  and  coagu- 
lates some  of  the  protein  constituents  making  it  less  digestible.  The 
desired  end  may  be  reached  by  using  a  lower  temperature  for  a  longer 
period  of  time,  and  the  method  generally  recommended  is  to  heat  the 
milk  to  60°  to  65°  for  thirty  minutes.  This  heating  is  sufficient  to 
render  harmless  any  pathogenic  organisms  likely  to  be  present  in  the 
milk,  without^the  objectionable  features  attendant  on  heating  to  a 
higherjdegree. 

Condensed  Milk. — It  is  commonly  stated  that  Gail  Borden  invented 
the  process  for  preparing  condensed  milk,  in  1856.  Previous  to  this, 
however,  milk  had  been  condensed  in  France,  England  and  Germany 
as  early  as  1825  to  1835.  While  he  cannot,  therefore,  be  called  the 
inventor  of  condensed  milk,  to  Borden  belongs  the  credit  of  having 
first  prepared  it  by  a  rational  process,  and  in  a  practicable  form. 

In  the  manufacture  of  condensed  milk,  good  fresh  milk  is  evaporated 
in  a  vacuum  pan  similar  to  those  used  in  sugar  factories,  at  a  tempera- 
ture of  40°  to  50°  until  the  volume  is  reduced  to  a  little  more  than  half, 
cane  sugar  being  added  so  that  the  finished  condensed  milk  usually 
contains  40  per  cent  cane  sugar.  The  evaporation  must  be  conducted 
with  great  care,  otherwise  the  lactose  crystallizes  out,  and  this  causes 
the  product  to  feel  "sandy"  to  the  tongue.  When  the  evaporation 
of  the  milk  is  complete,  the  yellowish  white  syrup  is  sealed  up  in  tins 


532  MICROBIOLOGY   OF   FOODS 

which  hold  about  450  g.,  and  this  quantity  is  equivalent  to  about  i  J^  1. 
of  normal  milk.  The  addition  of  cane  sugar  acts  as  a  preservative, 
and  although  the  finished  product  may  contain  some  living  organisms, 
it  is  said  to  keep  indefinitely  if  unopened,  and  will  even  keep  for  a 
number  of  days  after  opening.  Occasional  losses  do  occur  by  spoilage 
of  the  finished  product,  either  from  the  growth  of  occasional  types 
of  bacteria  tolerant  of  the  high  percentage  of  cane  sugar,  or  from 
yeasts. 

PROCESSING  AND  STERILIZATION 

ECONOMIC  CONSIDERATIONS. — For  certain  classes  of  food  products, 
pasteurization  is  widely  applicable,  and  is  of  great  value  from  an 
economic  standpoint.  Preservation  by  pasteurization  is  at  best,  how- 
ever, temporary.  Bacterial  spores  are  certain  to  be  present  on  many 
kinds  of  foods,  and  these,  unharmed  by  pasteurizing  temperatures, 
develop  vegetative  cells,  and  spoilage  occurs. 

For  permanent  preservation  therefore,  a  higher  temperature  and 
longer  periods  of  time  must  be  adopted.  The  final  heating  of  canned 
foods  for  permanent  preservation  has  formerly  been  termed  steriliza- 
tion. In  the  light  of  recent  researches,  this  terminology  must  be 
modified. 

As  stated  in  a  previous  paragraph,  it  has  been  shown  by  several 
investigators  that  canned  food  may  keep  for  a  long  period  of  time  al- 
though not  in  a  sterile  condition,  possibly  containing  viable  spores  of 
bacteria.  These  spores  are  unable  to  grow  in  the  food  due  to  the  ab- 
sence of  oxygen.  The  principle  employed  in  the  canning  of  food 
to-day  is  the  same  as  that  of  Appert  over  100  years  ago.  Although  he 
knew  nothing  of  microorganisms  or  their  relation  to  the  spoilage  of  food, 
Appert's  experiments  taught  him  that  not  only  must  the  food  to  be 
conserved  be  heated  thoroughly,  but  it  must  also  be  so  sealed  as  not  to 
allow  air  to  enter  the  container. 

With  the  development  of  knowledge  of  microbiology  it  was  con-' 
sidered  that  the  success  of  the  process  did  not  depend  so  much  upon 
keeping  out  the  air,  but  upon  keeping  out  organisms  which  might  be 
carried  in  the  air.  This  is  true  provided  the  canned  food  is  sterilized. 
As  recently  proven,  however,  commercially  canned  foods  may  keep 
perfectly  although  not  actually  sterile,  provided  a  vacuum  is  established 
in  the  can  thereby  preventing  the  viable  spores  of  bacteria  from 
developing. 


HEAT  IN  THE  PRESERVATION  OF  FOOD  PRODUCTS     533 

The  final  heating  of  the  food  in  the  cans  is  more  properly  called 
processing,  a  term  which  has  long  been  in  use  by  the  commercial  canner, 
and  under  present  methods  of  processing,  the  vacuum  in  the  can  appears 
to  be  as  essential  to  the  preservation  of  the  food  as  the  heating  itself. 

Conditions  arising  as  a  result  of  the  World  War  have  stimulated  a 
great  deal  of  investigation  by  various  workers  to  establish  the  tempera- 
tures and  length  of  time  necessary  for  processing  to  secure  actual 
sterility  of  food  products,  this  condition  being  very  properly  the  ulti- 
mate aim  of  the  packer. 

SPECIFIC  APPLICATION. — For  the  preservation  of  fruit  juices  and 
fermented  products,  pasteurization  is  much  more  extensively  used 
than  processing  at  a  higher  temperature.  If  too  high  a  temperature  is 
employed  for  fruit  juices,  certain  compounds  of  agreeable  taste  and 
aroma  are  destroyed,  with  a  consequent  deterioration  in  the  flavor  of 
the  product.  Fruit  juices  may  be  sterilized  by  heating  at  a  low  tempera- 
ture for  a  period  of  time  on  each  of  several  successive  days. 

The  method  of  Appert  has  its  widest  application  in  the  conservation  of  fruits, 
vegetables,  meats  and  fish.  Whatever  modifications  are  made  in  the  handling  of  the 
different  classes  of  foods,  the  essentials  are  the  same.  The  raw  material,  after 
thorough  cleaning  and  removal  of  waste  if  any,  is  filled  into  the  cans  and  submitted 
to  the  heating  process,  the  degree  of  heat  and  time  of  processing  varying  with 
different  foods.  With  a  few  exceptions,  notably  asparagus,  vegetables  are  im- 
proved by  heating  above  the  boiling  point.  With  fruits  the  reverse  is  true,  the  con- 
servation of  flavor  being  best  at  as  low  a  temperature  as  is  practicable  to  be  employed 
to  properly  preserve  them  from  spoilage.  Briefly  the  methods  employed  in  canning 
some  foods  follows: 

Meat. — The  canning  of  meat  for  interstate  commerce  is  under  Government 
supervision.  No  meat  may  be  used  which  has  not  undergone  inspection,  the  plants 
must  comply  with  certain  prescribed  regulations,  and  the  methods  be  approved. 
This  is  the  only  line  of  canning  under  inspection.  It  practically  limits  the  canning 
of  meat  to  the  large  slaughter  houses,  or  to  companies  purchasing  only  inspected 
products,  and  having  inspectors  in  their  plants. 

In  the  meat-canning  industry,  lean  meat  is  largely  selected  for  two  reasons. 
Fat,  well-finished  carcasses  bring  a  better  price  when  offered  for  sale  in  the  fresh  con- 
dition; and  in  the  second  place,  lean  meat  has  a  better  appearance  in  the  canned 
state  than  fat  meat.  The  selected  meat  is  cut  into  pieces  of  approximately  from 
i  to  4  pounds  in  weight,  according  to  the  size  of  the  tins  in  whick  it  is  to  be  pre- 
served. The  pieces  are  cut  as  nearly  as  practicable  the  same  size,  not  only  for  pur- 
poses of  appearance  in  the  cans  when  opened,  but  also  that  the  heating  process  may 
be  more  uniformly  carried  out.  If  the  pieces  were  of  different  sizes,  the  smaller 
ones  would  become  thoroughly  cooked  and  disintegrated  before  the  larger  ones  were 
sufficiently  processed. 


534  MICROBIOLOGY   OF   FOODS 

After  the  pieces  have  been  selected  and  dressed  they  are  parboiled  before  being 
placed  in  the  containers,  the  time  ranging  from  eight  to  twenty  minutes,  according  to 
the  size  of  the  pieces.  The  object  of  parboiling  is  to  secure  the  shrinkage  which 
always  takes  place  on  heating.  Meats  put  into  tins  in  the  fresh  state  and  processed 
shrink  to  about  two-thirds  of  their  original  volume.  When  the  meat  is  put  directly 
into  boiling  water,  there  is  less  loss  of  protein  than  when  placed  in  cold  water  and 
heated  gradually.  During  parboiling,  the  meat  loses  about  i  per  cent,  of  the  pro- 
tein content,  about  one-third  of  the  total  meat  bases,  and  50  per  cent,  of  the  mineral 
matter. 

This  shrinkage  by  parboiling  tends  to  make  a  more  concentrated  article,  thus 
favoring  transportation,  and,  pound  for  pound,  the  nutritive  value  is  not  lowered. 
Practically,  the  nutritive  value  of  a  pound  of  properly  canned  beef  is  about 
one-third  greater  than  that  of  i  pound  of  fresh  beef  of  the  same  kind.  After  par- 
boiling, the  meat  is  placed  in  tins  and  a  quantity  of  meat  jelly  is  added  to  prevent 
the  meat  from  adhering  to  the  tin  in  spots,  and  also  to  give  it  a  better  appearance. 

Some  meats  are  partially  cured  before  canning,  as  corned  beef.  Sausages,  and 
minced,  devilled,  and  potted  meats  are  cooked  and  run  through  meat  cutters  or 
grinders.  These  products  are  generally  made  from  meat  trimmings  and  pieces  too 
small  to  use  in  the  regular  way.  Some  of  these  contain  mixtures  of  meats,  some 
cereal,  and  others  spices.  The  packing  of  chicken,  turkey,  and  game  follows  the 
general  routine  of  meat  packing. 

Fish. — The  process  of  fish  canning  does  not  differ  materially  from  that  of  other 
meats.  On  account  of  its  proneness  to  rapid  decomposition,  especial  care  must  be 
observed  that  the  fish  are  in  a  perfectly  fresh  state  before  canning,  and  that  the 
processing  be  most  thorough.  The  salmon  is  preeminently  the  sea-food  in  cans  in 
this  country,  the  value  of  the  pack  being  nearly  equal  to  all  other  sea-foods  com- 
bined. Further,  salmon  is  the  principal  fish  for  the  preservation  of  which  depend- 
ence is  placed  on  sterilization  alone,  most  fish  being  preserved  by  other  methods. 

Vegetables  and  Fruits.  Corn. — Sweet  corn  only  is  used.  The  young  tender  ears 
of  sweet  corn  are  picked  from  the  stalk,  preferably  in  the  early  morning,  keeping  the 
husks  on,  and  are  taken  in  this  condition  to  the  factory.  They  are  husked  and  the 
silks  removed  and  passed  through  machines  with  sets  of  knives  which  cut  the  grains 
evenly  from  the  cob,  care  being  observed  not  to  cut  the  corn  so  closely  as  to  cut  off 
particles  of  the  cob  with  the  corn.  In  some  cases  the  cobs  are  next  passed  through 
scrapers  which  remove  the  small  tips  adherent  to  the  cob.  After  the  corn  is  cut  it  is 
run  through  a  cleaner  which  removes  bits  of  cob,  husk,  and  silk.  It  is  then  passed 
to  a  mixer  and  the  proper  amount  of  water,  bearing  sugar  and  salt  in  solution  is 
thoroughly  stirred  through  the  mass.  It  is  then  run  into  the  filler  and  cooker.  Most 
of  the  operations  are  done  by  machinery,  and  the  different  processes  follow  each  other 
in  such  rapid  succession  that  from  the  time  the  ear  goes  into  the  husking  machine 
until  the  corn  is* in  the  can,  sealed,  and  ready  for  the  retort  may  not  be  more  than 
fifteen  minutes. 

Peas. — In  the  pea-canning  industry  the  vines  are  cut  with  a  mower  or  a  special 
pea  harvester,  loaded  onto  racks  and  hauled  to  the  vining  machines.  The  viner  is  a 
machine  consisting  of  an  outer  and  an  inner  cylinder  revolving  in  opposite  directions, 


HEAT   IN   THE   PRESERVATION   OF   FOOD    PRODUCTS  535 

the  inner  one  bearing  paddles  or  beaters  so  arranged  that  as  the  vines  pass  through 
the  machine  the  paddles  break  open  the  pods.  As  the  peas  are  thrown  out,  they 
pass  through  perforations  in  the  outer  cylinder,  while  the  vines  are  discharged  at  the 
opposite  end.  The  peas  are  run  through  a  fanning  mill  to  blow  out  bits  of  stems, 
leaves,  and  pods  after  which  they  are  washed  to  remove  all  dirt  and  also  the  mucous 
substance  from  the  surface  thus  insuring  a  clearer  liquor  in  the  can.  The  peas  are 
next  passed  through  a  sizer,  which  separates  them  into  five  sizes  or  grades.  Some 
peas  are  packed  ungraded  and  the  proportion  thus  packed  is  increasing.  The  peas 
are  next  blanched  to  drive  water  into  them  so  that  all  will  be  tender.  The  time 
of  blanching  varies  from  one-half  to  five  or  more  minutes,  large  mature  peas  requiring 
more  time  for  the  blanching  than  young  tender  ones.  The  peas  are  then  filled  into 
cans  by  machines  which  deliver  exact  quantities  together  with  the  necessary  brine 
after  which  they  are  processed. 

Fruits. — The  essentials  in  the  canning  of  fruits  do  not  differ  from  those  for 
vegetables.  Stone  fruits  may  be  canned  either  with  or  without  the  pits.  In  the 
case  of  such  fruits  as  cherries,  or  other  acid  fruits,  the  tin  is  coated  on  the  inside 
with  a  lacquer  or  enamel  which  protects  the  tin  from  erosion  by  the  action  of  the 
acid  juices.  The  time  and  temperature  of  processing  fruits  is  usually  less  than 
that  required  for  vegetables,  for  the  reason  that  in  the  presence  of  the  fruit  acids 
the  organisms  are  more  easily  destroyed  than  in  foods  in  which  acids  are  not  present. 


CONTROLLING  FACTORS  IN  SUCCESSFUL  CANNING 

CLEANLINESS. — Too  much  emphasis  could  hardly  be  placed  upon 
the  importance  of  cleanliness  throughout  the  whole  preserving  process, 
and  especially  in  the  preparation  of  the  food  for  preserving.  Vege- 
tables that  have  come  into  contact  with  the  soil  are  pretty  certain  to 
harbor  many  spores  of  bacteria,  and  if  as  many  of  these  are  removed 
as  possible  by  a  thorough  preliminary  cleansing,  processing  may  be 
effected  with  greater  ease  and  certainty.  The  necessity  of  cleanliness 
on  the  part  of  factory  employees  is  needful  only  of  mention,  not  only 
from  the  esthetic  standpoint,  but  also  from  that  of  good  health. 

THE  SOUNDNESS  OF  RAW  MATERIAL. — The  necessity  of  sound  and 
wholesome  raw  material  is  fully  as  great  as  that  of  cleanliness  in 
handling.  Foods  are  never  better  than  when  they  are  fresh.  It  makes 
no  difference  how  long  nor  by  what  method  they  may  be  cooked,  the 
quality  cannot  be  bettered,  and  if  food  is  unsound  when  put  into  the 
containers  for  canning,  it  will  never  be  wholesome  for  food;  and  this 
fact  is  equally  true  whether  the  unsoundness  is  the  result  of  diseased 
conditions  of  meats,  fruits,  or  other  products,  or  whether  it  is  due  to 
ordinary  decay. 


536  MICROBIOLOGY   OF  -FOODS 

WATER  SUPPLY. — Another  essential  for  the  success  of  the  canner  is 
an  ample  supply  of  pure  water.  It  is  a  well-known  bacteriological 
fact  that  outbreaks  of  spoilage  have  occurred  in  canneries  which  could 
be  traced  to  organisms  getting  into  the  goods  from  the  water  supply. 

RECEPTACLES. — The  commercial  canner  recognizes  two  essentials  for 
suitable  containers  for  his  goods.  First,  they  must  be  tight,  both  to 
prevent  the  escape  of  the  contained  material  and  the  entrance  of  con- 
taminating organisms.  Second,  they  must  be  of  a  material  which  will 
withstand  erosion  or  corrosion  for  a  reasonable  length  of  time,  without 
giving  up  any  notable  quantity  of  foreign  material  to  the  food  with 
which  they  may  be  in  contact.  Glass  is  most  satisfactory  from  this 
consideration,  but  for  reasons  previously  stated  it  is  impracticable  for 
use  on  a  commercial  scale.  The  difficulty  from  erosion  in  tin  cans  has 
been  largely  overcome  by  the  use  of  enamelled  cans  as  mentioned  above. 

DEGREE  OF  HEAT  REQUIRED.  *  Factors  in  Processing. — The  heating 
of  food  products  after  placing  in  the  containers  is  termed  processing 
by  the  commercial  canner,  and  he  appreciates  fully  that  upon  the  care 
with  which  the  processing  is  done  depends  the  success  of  the  entire  pack. 

The  several  factors  which  enter  into  the  successful  processing 
of  foods,  either  in  commercial  or  home  canning  may  be  enumerated  as 
follows :  (a)  length  of  time,  (b)  number  and  resistance  of  spores  present 
in  the  material,  (c)  size  of  container,  (d)  consistency  of  contents  of  the 
container,  (e)  initial  temperature,  (/)  agitation  of  the  container  during 
processing. 

Length  of  Time  Required. — Fig.  152  gives  the  curves  showing  the 
time  necessary  at  various  temperatures  to  destroy  the  spores  of  three  of 
the  most  resistant  organisms  found  in  canned  foods  when  about  fifty 
thousand  spores  per  c.c.  are  present.  It  will  be  noted  that  a  drop  of  ten 
degrees  Centigrade  necessitates  about  ten  times  as  long  for  the  destruc- 
tion of  the  spores.  For  instance  in  organism  No.  26  six  minutes  at 
i25°C.  are  necessary  and  about  sixty-five  minutes  at  115°. 

Number  and  Resistance  of  Spores. — Fig.  1 53  shows  the  influence  of 
the  number  of  spores  on  the  processing  time.  It  will  be  noted  that 
organism  "C"  when  about  twenty  spores  were  present  required  twelve 
minutes  for  their  destruction;  when  about  fifty  thousand  spores  were 
present,  sixty  minutes  were  necessary.  In  other  words,  at  a  tempera- 
ture of  n5°C.  nearly  six  times  as  long  was  found  to  be  necessary 

*  The  author  is  indebted  for  charts  and  data  to  Dr.  W.  D.  Bigelow,  Chief  Chemist  of  the 
National  Canners  Association. 


HEAT   IN   THE   PRESERVATION    OF   FOOD    PRODUCTS  537 


L  (*a?r) 

s 

3  /?0K 

K  <2w95' 


?   //<£?"<r 

|J4    C330V} 


76O  OOO    040   S&O  &2O 
77/W  //(/  /V//W7Z& 

'    FIG.  152. — Influence  of  temperature  on  sterilizing  time 


FIG.  153. — Influence  of  number  of  spores  on  sterilizing  time. 


538 


MICROBIOLOGY   OF   FOODS 


in  the  presence  of  fifty  thousand  spores  as  in  the  presence  of  about 
twenty  spores. 

Size  of  Container. — Assuming  the  same  food  product,  it  naturally 
follows  that  a  large  can  would  require  a  longer  time  for  processing  than 
a  small  one,  the  rapidity  of  heat  penetration  varying  according  to  the 
size  of  the  can. 

Consistency  of  Contents  of  the  Container. — Consistency  is  expressed 
in  the  way  it  is  determined  when  a  can  is  examined  after  it  has  been 
packed,  that  is,  in  terms  of  drained  solids.  A  can  containing  twenty- 
seven  ounces  of  drained  solids  will  require  a  longer  processing  time  than 
the  same  size  can  containing  only  eighteen  ounces  of  drained  solids.  Or, 
to  apply  the  principle  in  another  way,  a  product  such  as  peas  or  carrots 
will  require  less  time  for  heat  penetration  than  corn  or  spinach  in  which 
the  food  is  packed  more  closely  in  the  can. 

Initial  Temperature. — Foods  filled  into  the  cans  hot  can  be  success- 
fully processed  in  a  shorter  time  than  if  packed  cold.  This  is  especially 
true  in  the  case  of  such  goods  as  corn  in  which  the  heat  penetration  is 
slower  than  with  some  other  products  such  as  peas. 


FIG.    154. — Tomatoes    in   no.   3   cans — Influence  of  speed  of  rotation  on     heat 

penetration. 

Agitation  of  the  Container  during  Processing. — This  factor  can  best 
be  explained  by  Fig.  154  which  shows  the  influence  on  heat  penetration 
of  rotating  a  can  of  tomatoes  while  it  is  being  processed.  The  can 
represented  by  curve  "B"  was  processed  in  the  ordinary  steel  retort 
without  rotating.  The  can  in  curve  "A"  was  processed  continuously 


HEAT  IN  THE  PRESERVATION  OF  FOOD  PRODUCTS     539 

at  eleven  revolutions  per  minute.  The  cans  in  the  curves  marked 
"C"  and  "D"  were  processed  according  to  the  practice  of  one  of  the 
typical  commercial  cookers.  It  will  be  noticed  that  whereas  the  center 
of  a  #3  can  of  tomatoes  required  about  twenty-eight  minutes  to  reach 
the  temperature  of  180°  without  rotation,  it  reached  the  same  tempera- 
ture in  three  or  "four  minutes  with  rotation.  In  general  it  may  be  said 
that  a  product  consisting  of  solid  particles  surrounded  by  a  relatively 
clear  liquor  heats  to  the  center  of  the  can  almost  as  quickly  as  if  the 
particles  were  not  there,  that  is,  almost  as  quickly  as  water  in  a  still 
retort  so  that  agitation  is  not  of  particular  value  in  such  products. 
A  product  of  a  viscous  nature  which  interferes  with  convection  cur- 
rents, however,  heats  to  the  center  of  the  can  much  more  quickly  if 
there  is  some  form  of  agitation  which  supplements  the  convection 
.currents.  By  rotation  as  mentioned  above  is  meant  the  rotating  of  a 
can  on  its  axis  as  done  by  some  of  the  commercial  cookers  now  widely 
used  with  tomatoes  and  some  other  products. 

HOME  CANNING  OF  FOODS 

The  successful  canning  of  foods  in  the  home  depends  upon  the  same 
principles  as  those  employed  in  commercial  canning,  namely,  cleanli- 
ness, soundness  of  raw  material,  and  thorough  processing.  Aside  from 
the  universally  used  open  kettle  method  of  handling  the  foods,  two  other 
methods  are  now  widely  used:  the  cold-pack  method,  and  the  vacuum- 
seal  method.  In  the  cold-pack  method,  the  products  are  cleaned, 
blanched  by  dipping  in  hot  water,  and  immediately  packed  into  the 
glass  jars  or  other  con  tamers.  To  fruits  hot  syrup  may  be  added;  to 
vegetables  and  greens  hot  water  and  a  little  salt  is  added.  Preserva- 
tion of  fruits  and  some  vegetables  may  be  effected  by  the  intermittent 
or  fractional  method  of  heating  on  each  of  three  successive  days, 
the  time  of  heating  depending  on  the  nature  of  the  product  and  the 
size  of  the  container  as  described  above.  The  extra  time,  labor,  and 
fuel  required  for  this  method  makes  it  impractical  where  a  large  amount 
of  canning  is  to  be  done.  Furthermore,  as  Bigelow  and  Estey  have 
shown,  foods  may  carry  organisms  the  spores  of  which  are  not  destroyed 
by  fractional  processing,  and  these  may  cause  spoilage  of  the  canned 
product.  The  more  common  method  of  home  canning  is  to  process  the 
food  in  one  period  by  immersion  of  the  containers  in  water  which  is  then 
brought  up  to  the  boiling  point;  or  by  the  use  of  steam  pressure  outfits 


540  MICROBIOLOGY   OF   FOODS 

of  which  the  market  affords  several  types.     These  operate  on  the  same 
principle  as  the  laboratory  autoclav  or  the  commercial  canner 's  retort. 

SPOILAGE  or  CANNED  FOODS 

MICROBIAL  changes  occur  when  the  goods  have  not  been  proc- 
essed at  a  temperature  sufficiently  high  to  destroy  all  the  organisms 
present  in  the  uncooked  food,  or  when  a  vacuum  has  not  been  estab- 
lished. In  some  instances,  the  organisms  decompose  the  contents  of 
the  can  with  formation  of  gas,  causing  bulging  of  the  ends  of  the  cans 
sometimes  to  the  point  of  bursting  at  the  seams.  Such  cans  are  desig- 
nated at  the  factory  as  "swells."  In  other  instances,  the  bacteria 
cause  an  acid  fermentation  with  consequent  souring  of  the  contents. 
The  canner  terms  such  cans  "flat  sours." 

DETECTION  OP  SPOILED  GOODS. — In  rases  of  spoilage  accompanied 
by  gas  production,  detection  of  the  spoiled  cans  is  easy  from  the 
bulged  appearance  of  the  ends  of  the  cans.  On  account  of  the  exhaus- 
tion of  air  from  the  cans  previous  to  processing,  the  ends  of  sound  cans 
should  be  slightly  concave.  If  the  ends  of  the  cans  are  convex,  it 
indicates  some  abnormal  condition  of  the  contents  and  such  cans  should 
be  rejected.  In  the  case  of  sours,  detection  is  not  so  easy.  The  can 
may  appear  normal,  and  there  may  be  no  change  in  the  contents  appar- 
ent to  the  eye  on  opening  the  can.  Taste,  however,  reveals  a  more  or 
less  pronounced  disagreeable  acid  flavor.  Canned  meats,  fish,  or  crus- 
taceans are  likewise  liable  to  spoilage  if  the  processing  has  been  imper- 
fectly carried  out.  In  these  goods  the  change  is  generally  accompanied 
by  gas  production,  hence  detection  is  easy  because  of  the  swelled 
appearance  of  the  cans. 

If  gas  production  is  present,  or  there  is  an  odor  resembling  rancid 
cheese,  or  if  the  contents  appear  mushy  or  disintegrated,  in  no  case 
should  the  contents  of  the  can  be  tasted  to  see  if  it  is  spoiled,  as  these 
conditions  indicate  spoilage  by  B.  botulinus,  and  the  toxin  of  this 
organism  may  prove  fatal  in  the  smallest  traces. 

DISPOSAL  OF  FACTORY  REFUSE 

The  disposal  of  factory  refuse  has  at  times  been  a  serious  problem  for  the 
commercial  canner.  Of  late  years  methods  have  been  devised  for  utilizing  much 
of  the  material  that  formerly  was  allowed  to  accumulate  about  the  factory 
in  fermenting  heaps  to  the  extent  of  sometimes  becoming  a  nuisance  to  the  neigh- 
borhood. 


HEAT  IN   THE   PRESERVATION   OF   FOOD   PRODUCTS  54! 

At  pea  canneries  several  methods  of  utilizing  the  vines  are  in  use.  They  may 
be  converted  into  silage,  either  by  putting  into  silos  or  stacking  in  large  stacks. 
In  some  sections  the  vines  are  cured  for  hay.  They  are  also  valuable  as  a  fertilizer. 

Corn  husks  and  cobs  are  also  used  for  silage.  Experiments  were  made  by  the 
United  States  Department  of  Agriculture  in  regard  to  the  feasibility  of  using  the 
refuse  from  the  canning  of  corn  for  the  production  of  alcohol.  It  was  found  that,  on 
account  of  the  expensive  machinery  and  apparatus  required  in  the  manufacture, 
a  small  factory  could  not  profitably  utilize  the  corn  waste  for  alcohol.  It  was 
shown  that  where  several  factories  were  located  within  a  short  radius  of  each  other, 
by  shipping  their  waste  to  a  central  plant,  it  might  be  used  up  to  advantage. 

Apple  cores,  "chops"  and  peelings  are  usually  either  used  for  vinegar  making,  or 
are  made  up  into  apple  jelly.  From  one  factory  visited  by  the  writer,  the  apple 
cores  and  peelings  were  dried,  baled,  and  shipped  to  Europe,  "to  be  made  up  into 
champagne." 

Peach  pits  are  sometimes  sold  to  nurserymen  for  seed.  Sometimes  the  pits  are 
cracked  and  the  meats  used  for  almond  meats  and  also  oil.  In  many  factories,  no 
use. is  made  of  the  peach  stones. 

In  the  classes  of  foods  in  which  the  waste  is  not  large,  the  refuse  is  hauled 
away  to  a  dumping  ground  near  the  factory,  or  is  taken  away  by  farmers  for  its 
manurial  value. 


CHAPTER  III* 
THE  PRESERVATION  OF  FOOD  BY  COLD 

INTRODUCTION 

In  recent  times  cold  storage  has  become  of  very  great  importance 
in  the  preservation  of  perishable  food  stuffs,  and  foods  preserved  by 
cold  usually  command  a  higher  market  price  than  those  preserved  by 
other  methods.  This  is  probably  due  primarily  to  the  fact  that  the 
general  appearance  of  refrigerated  food  resembles  that  of  the  perfectly 
fresh  article,  in  many  instances  very  closely.  Moreover,  in  many 
instances  cold  storage,  for  a  reasonable  length  of  time,  preserves  not 
only  the  appearance  and  the  nutritive  value,  but  also  the  chemical 
composition  and  even  the  delicate  flavors  of  the  original  articles,  so 
important  in  determining  market  value.  The  great  economic  impor- 
tance of  this  industry  is  at  once  apparent,  for  it  aims  to  preserve  un- 
changed the  over-abundance  of  one  locality  for  transportation  to 
another  and  the  over-production  of  one  season  of  the  year  for  subse- 
quent use. 

THE  EFFECTS  OF  REFRIGERATION  UPON  FOODS  IN  GENERAL 

The  decomposition  of  foods  depends  upon  the  activity  of  their  own 
intrinsic  enzymes  to  some  extent,  but  more  especially  upon  the  activity 
of  foreign  microorganisms — bacteria,  yeasts  and  molds.  Cold  acts  as 
a  preservative,  not  by  destroying  these  microbes,  but  by  retarding  or 
inhibiting  their  activity.  In  general,  cold  not  only  retards  the  growth 
of  the  microorganisms  but  delays  their  death  also,  tending  to  preserve 
them  as  well  as  the  food  unchanged. 

In  discussing  the  refrigeration  of  foods  we  may  consider  three  periods 
of  treatment,  (i)  the  removal  of  the  heat  or  chilling  of  the  food,  (2)  the 
prolonged  storage  at  low  temperature,  (3)  the  subsequent  warming  of 
the  food  before  sale  or  consumption. 

•  Prepared  by  W.  J.  MacNeal. 

542 


THE  PRESERVATION  OF  FOOD  BY  COLD          543 

CHANGES  DURING  CHILLING. — The  period  of  cooling  is  a  relatively 
short  one,  varying  from  a  few  hours  to  a  few  days  in  length.  The  chief 
physical  change  is  the  intentional  removal  of  heat  by  conduction  and 
convection,  but  there  is  usually  also  some  loss  of  water  by  evaporation. 
If  cooled  to  a  sufficient  degree  the  water  content  of  the  food  may 
crystallize,  altering  to  a  considerable  extent  the  physical  structure  of 
the  food  substance  (frozen  food).  Most  foods  are  either  actually 
living  when  chilling  begins,  or  they  are  only  recently  dead  and  various 
chemical  changes  due  to  intrinsic  enzymes  continue  at  a  diminishing 
rate  as  the  heat  is  removed.  Decomposition  changes,  due  to  microbes, 
may  also  be  in  progress  and  continue  during  the  process  of  chilling. 
At  this  time  the  microbes  living  in  the  cold-storage  chamber  gain  access 
to  the  newly  arrived  food  and  others  are  added  in  the  process  of  handling. 
The  extent  to  which  these  will  grow  and  multiply  depends  upon  their 
ability  to  flourish  under  the  storage  conditions.  In  general  the  bacteria 
which  flourish  at  ordinary  temperatures,  producing  the  familiar  decom- 
position of  the  particular  food,  are  greatly  retarded  in  their  activities 
and  other  kinds  of  microbes  outstrip  them  under  the  new  conditions. 
The  changes  taking  place  during  chilling  are  of  great  importance 
in  some  special  instances,  and  often  a  very  definite  procedure  must  be 
followed  to  obtain  a  satisfactory  result. 

CHANGES  DURING  STORAGE. — This  is  often  a  very  long  period  so 
that  causes  acting  very  slowly  may  ultimately  produce  marked  altera- 
tions. There  is  ordinarily  some  loss  of  water  by  evaporation,  as  well 
as  the  evaporation  or  diffusion  of  other  volatile  constituents,  some  of 
them  at  times  important  factors  in  the  flavor  of  the  food.  Other  vola- 
tile substances  may  be  absorbed  from  the  air  of  the  storage  room 
introducing  undesirable  odor  or  flavor.  The  chemical  changes  of  the 
chilling  period  continue  at  a  greatly  diminished  rate,  or  may  be  entirely 
inhibited  if  the  food  is  frozen.  The  behavior  of  the  microbic  content  of 
the  food  is  the  most  important  factor  to  be  considered  during  this 
period.  Besides  those  already  present,  various  other  microorganisms, 
bacteria,  yeasts  or  molds,  may  gain  access  to  the  food  from  time  to  time, 
either  from  the  circulating  air  or  by  contact  with  other  things.  The 
fate  of  the  implanted  microbes  will  depend  upon  their  nature  and  adap- 
tation to  the  conditions  existing  in  the  stored  food.  Many  of  them 
perish,  but  many  also  survive  the  entire  period  of  storage,  and  some  may 
actively  multiply.  There  can  no  longer  be  any  doubt  that  some  bac- 


544  MICROBIOLOGY   OF   FOODS 

teria  can  grow  at  the  temperature  of  zero,  and  many  kinds  multiply  at  a 
fraction  of  a  degree  above  that  point.  In  order  definitely  to  inhibit 
microbic  activity  the  food  must  be  frozen.  When  it  is  not  frozen, 
bacteria  continue  to  multiply  slowly  at  the  lowest  temperature  of 
storage,  and  small  variations  in  the  temperature  and  in  the  humidity 
of  the  atmosphere  serve  to  accelerate  their  activity.  Such  variations 
also  accelerate  diffusion  currents  in  the  food  substance  and  so  tend  to 
distribute  the  microorganisms  and  their  products.  The  extent  of  the 
resulting  chemical  changes  in  the  food  will  depend  upon  these  factors 
and  upon  the  nature  of  the  food,  the  temperature  and  the  length  of  the 
period  of  storage. 

CHANGES  AFTER  STORAGE. — This  is  a  relatively  short  period,  but  in 
many  instances  a  very  important  one  as  regards  change  in  the  food. 
If  warmed  too  rapidly,  vigorous  currents  may  be  set  up  in  the  food 
mass  by  the  great  difference  in  temperature  between  the  outer  portion 
and  the  interior,  serving  to  distribute  microorganisms  and  their 
products.  In  the  case  of  frozen  foods  rapid  warming  fails  to  restore 
the  original  physical  structure.  Dry  cold  foods  are  likely  to  condense 
moisture  from  the  warmer  atmosphere  unless  it  is  particularly  dry, 
and  this  condensed  water  becomes  another  cause  of  diffusion  currents. 
In  frozen  foods  the  water,  in  melting,  may  fail  to  reenter  the  food  struc- 
ture, and  exude  and  drip  away,  carrying  a  portion  of  the  soluble  con- 
stituents with  it.  At  this  time,  still  more  microbes  are  likely  to  be  added 
to  the  food,  and,  together  with  those  already  present,  they  multiply 
with  increasing  rapidity  as  the  temperature  rises.  As  they  may  al- 
ready be  pretty  well  distributed  throughout  the  mass  of  the  food,  the 
resulting  chemical  decomposition  is  the  more  rapid;  It  is  well  recog- 
nized that,  in  keeping  qualities,  foods  removed  from  cold  storage  are 
much  inferior  to  the  corresponding  fresh  foods. 

REFRIGERATION  OF  CERTAIN  FOODS 

MEAT,  FISH  AND  POULTRY. — Meat,  in  this  sense  the  flesh  of 
mammals,  is  preserved  by  cold  in  two  ways,  by  storage  above  the 
freezing-point  (chilled  meat)  and  by  storage  at  — 10°  to  —4°  (frozen 
meat).  Fish  and  poultry  are  usually  frozen  for  storage,  often  in  the 
undrawn  condition. 

Mammals  killed  for  chilled  or  for  frozen  meat  are  slaughtered  and 
carefully  dressed.  For  chilled  meat  the  temperature  is  reduced  by 


THE  PRESERVATION  OF  FOOD  BY  COLD          545 

storage  in  a  cold  air  chamber  to  about  +2°  in  forty-eight  hours,  and 
the  meat  is  stored  at  a  temperature  between  +1°  and  +2°.  Under 
these  conditions  the  enzymes  of  the  dead  flesh  continue  to  act  and 
bacterial  decomposition  proceeds  slowly,  bringing  about  a  process  of 
ripening  which,  up  to  a  certain  point,  improves  the  market  value  of  the 
flesh  by  making  it  more  tender  and  giving  to  it  a  more  desirable  flavor. 
The  extent  to  which  the  slow  bacterial  decomposition  may  proceed 
before  the  flavor  becomes  disagreeable  varies  with  different  tastes,  but 
in  general  the  beginning  of  proteolytic  change,  which  follows  after  the 
almost  complete  fermentation  of  the  muscle  sugar,  may  be  said  to  mark 
the  desirable  limit.  This  point  is  reached  in  from  a  week  to  three 
months,  depending  upon  the  condition  of  the  animal,  skill  and  care 
in  slaughter  and  dressing,  especially  the  extent  of  bacterial  contamina- 
tion at  this  time,  and  the  accurate  control  of  the  storage  conditions. 
Fresh  killed  beef  is  generally  regarded  as  quite  inferior  to  it.  In  the 
production  of  frozen  meat  the  carcasses  are  rapidly  chilled  in  an 
air  chamber  at  —20°,  where  the  meat  remains  until  frozen  solid. 
It  is  then  kept  at  a  temperature  below  —4°.  Freezing  produces  a 
marked  change  in  the  finer  physical  structure  of  the  meat,  as  the  water 
crystallizes,  leaving  the  protein  material,  with  which  it  was  formerly 
intimately  mixed,  in  a  shrunken  and  shriveled  state  between  the 
crystals.  Enzymic  and  bacterial  activities  are  practically  if  not  ab- 
solutely suspended  under  these  conditions,  and,  save  for  slight  surface 
evaporation,  such  meat  remains  unchanged  for  long  periods.  The 
subsequent  thawing  presents  certain  difficulties  and  requires  particular 
care.  If  warmed  very  slowly  the  melting  water  crystals  are  imbibed  by 
the  protein  material  and  the  original  structure  of  the  flesh  almost  com- 
pletely restored.  The  warmer  air  must  be  dry  and  must  be  kept  in 
motion  to  avoid  condensation  of  moisture  on  the  exterior  of  the  thaw- 
ing meat.  Bacterial  activity  is  likely  to  gain  considerable  headway 
during  this  process  and  the  penetration  of  the  microbes  into  the  flesh 
is  favored  by  the  diffusion  currents.  The  more  prolonged  the  warm- 
ing process,  the  greater  the  opportunity  for  bacterial  decomposition. 
Ordinarily,  to  avoid  this,  the  thawing  is  carried  out  rapidly  and  the 
finer  structure  of  the  meat  is  not  restored.  It  is  softer,  darker  and 
more  moist  than  fresh  or  chilled  meat,  and  usually  sells  at  a  lower 
market  price. 

It  is  preferable  that  frozen  meat  should  always  be  marketed  as  such 

35 


54$  MICROBIOLOGY   OF   FOODS 

and  should  come  into  the  hands  of  the  cook  while  still  frozen  hard.  As 
soon  as  portions  become  soft  they  should  be  cut  off  and  cooked.  The 
extensive  use  of  frozen  beef  during  the  war  has  proven  so  generally 
satisfactory  that  previous  prejudice  against  this  kind  of  meat  has  been 
largely  forgotten.  When  once  thawed  it  should  be  used  promptly. 

Fish  and  poultry  are  usually  frozen  for  storage.  As  these  foods  are 
especially  subject  to  rapid  objectionable  decomposition  changes  they  are 
rapidly  chilled  in  ice  water  or  by  packing  in  ice  immediately  after 
death,  and  are  frozen  as  quickly  as  possible.  During  storage  in  the 
frozen  condition  microbic  activity  is  suspended,  but  in  the  subsequent 
thawing  the  same  physical  and  biological  changes  occur  as  in  frozen 
meat.  When  fish  and  poultry  are  stored  in  the  undrawn  condi- 
tion there  is  an  abundant  supply  of  bacteria  at  hand  in  the  intestinal 
contents  ready  to  multiply  energetically  during  the  chilling  and  thawing 
stages.  It  would  appear  desirable  that  the  poultry  should  be  killed 
and  dressed  with  great  care  previous  to  freezing  and  that  the  period 
of  chilling  should  be  shortened  as  much  as  possible.  Practically,  how- 
ever, it  has  been  found  that  the  dressing  of  poultry,  as  ordinarily  done, 
previous  to  storage,  leads  to  such  an  extensive  soiling  of  the  edible  flesh 
of  the  birds  that  their  condition  at  the  end  of  the  storage  period  is  often 
less  satisfactory  than  that  of  undrawn  frozen  poultry,  not  only  in  gross 
appearance  but  also  in  respect  to  microbic  content  and  chemical  com- 
position. Most  frozen  poultry  is,  therefore,  stored  in  the  undrawn 
condition. 

The  tendency  of  such  food  to  undergo  decomposition  after  thawing 
should  be  clearly  recognized  and  prompt  cooking  at  once  after  softening 
should  be  insisted  upon.  Its  sale  as  fresh  or  as  chilled  food  is  a  fraud 
upon  the  purchaser.  In  fact  many  individuals  seem  to  be  pecu- 
liarly liable  to  suffer  digestive  disturbances  after  eating  frozen  poultry 
and  such  persons  should  avoid  its  use. 

The  nature  and  source  of  the  bacteria  which  produce  poisonous 
changes  in  poultry  are  not  definitely  known,  but  there  is  some  evidence 
indicating  that  they  belong  to  the  para-colon  group  and  that  they  are 
derived  from  the  intestinal  contents  of  the  fowls.  Smith  and  Ten- 
Broeck*  have  studied  a  typhoid-like  bacillus  found  in  the  intestinal 
contents  of  fowls.  This  organism  produces  a  poison  which  is  only 

*  Smith    and    TenBroeck,   Journal   of    Medical    Research,   Jan.,    1915,   Vol.  31,  No.  3,  pp. 
523-546. 


THE  PRESERVATION  OF  FOOD  BY  COLD          547 

partially  destroyed  by  boiling  for  15  minutes.  It  may  be  suggested 
that  organisms  of  this  type  existing  in  frozen  poultry  might  well  account 
for  poisonous  effects  produced  by  the  flesh  after  roasting  or  boiling. 

EGGS. — The  cold  storage  of  eggs  is  an  industry  which  has  attained 
large  proportions  in  recent  years.  A  very  constant  storage  temperature 
between  +0.5°  and  -fi°  is  essential  for  the  best  results.  The  hu- 
midity of  the  atmosphere  is  also  of  very  great  importance,  as  a  dry  air 
causes  extensive  evaporation  from  the  egg  and  a  too  moist  air  favors  the 
development  of  microorganisms  on  the  exterior  of  the  shell  and  the 
absorption  of  their  products  and  even  their  penetration  into  the  egg. 
A  constant  humidity  of  70  per  cent  saturation  has  been  found  to  be  the 
best.  Storage  at  this  temperature  and  humidity  greatly  retards  the 
growth  of  microorganisms  and  definitely  inhibits  the  ordinary  putre- 
faction of  eggs.  The  activity  of  the  intrinsic  enzymes  of  the  egg  are 
not  necessarily  inhibited  by  this  temperature,  nor  is  the  growth  of  all 
microorganisms  prevented.  Unquestionably  there  is  a  marked  differ- 
ence between  the  ordinary  cold-storage  egg  and  the  strictly  fresh  egg, 
but  to  what  extent  this  deterioration  may  be  due  to  errors  in  storage, 
such  as  inaccurate  control  of  temperature  and  humidity,  use  of  odorif- 
erous crates  for  packing,  decomposition  changes  previous  to  storage, 
too  rapid  chilling  of  the  eggs,  or  too  rapid  warming  of  them  after 
removal  from  storage,  and  to  what  extent  it  is  inherent  in  the  most 
perfect  cold-storage  procedure,  is  still  somewhat  uncertain.  Doubt- 
less a  certain  amount  of  deterioration,  especially  the  loss  of  the  peculiar 
flavor  of  the  fresh  egg,  is  unavoidable  in  any  method  of  prolonged 
storage.  The  discrimination  in  price  in  favor  of  new-laid  eggs  in  the 
market  is  an  indication  of  difference  in  actual  value,  and  the  sale  of 
cold-storage  eggs  for  new-laid  or  strictly  fresh  eggs  is  generally  recog- 
nized as  a  fraud  by  the  purchaser.  The  cold-storage  egg  is  nevertheless 
a  very  valuable  food  and  the  economic  importance  of  saving  the  over- 
abundant supply  produced  during  the  spring  for  use  during  the  colder 
season  of  the  year  makes  this  industry  a  great  benefaction  to  the  public. 
Suitable  regulation  may  be  expected  to  remove  its  objectionable 
features. 

MILK  AND  BUTTER. — Milk  as  ordinarily  sold  at  retail  is  not  subject 
to  sufficient  seasonal  change  in  market  price  to  make  its  prolonged 
storage  advisable.  But  milk  is  so  rapidly  changed  by  bacterial  activity 
at  ordinary  temperatures  that  efficient  dairy  methods  necessarily  in- 


548  MICROBIOLOGY   OF   FOODS 

elude  prompt  cooling  of  the  milk  after  it  is  drawn  from  the  animal 
and  the  maintenance  of  a  low  temperature  until  it  is  delivered  to  the 
consumer.  At  the  low  temperature  bacteria  slowly  multiply,  unless 
the  milk  is  actually  frozen,  but  at  a  temperature  slightly  above  the 
freezing-point  very  clean  milk  may  be  kept  in  perfect  condition  for  a 
week,  and  it  may  be  kept  sweet  for  several  weeks.  Refrigeration  of 
milk  cannot  compensate  for  unhealthy  animals  producing  it,  nor  for 
careless  and  uncleanly  methods  of  handling.  The  cold  does  not 
destroy  the  microbes  in  the  milk  but  only  retards  their  multiplication 
and  chemical  activity.  In  practice,  especially  in  the  transportation  of 
milk  into  large  cities,  it  is  frequently  most  economical  to  freeze  the  milk 
and  trust  to  insulation  and  the  latent  cold  in  the  ice  to  maintain  a  low 
temperature  during  transportation.  Such  milk  should  arrive  at  its 
destination  in  a  partly  frozen  condition. 

The  cold  storage  of  butter  is  essential  even  when  it  is  kept  for  only 
short  periods,  and  the  seasonal  variation  in  price  is  sufficient  to  warrant 
its  storage  from  summer  to  winter.  The  keeping  qualities  of  butter 
depend  upon  many  factors,*  and  the  most  efficient  cold  storage  cannot 
compensate  for  previous  deficiencies.  In  refrigerated  butter  there  is  a 
gradual  diminution  in  the  number  of  living  bacteria,  with  possibly 
a  multiplication  of  a  few  particular  kinds.  There  is  a  slow  increase  in 
acidity.  In  frozen  butter  the  bacterial  content  and  the  chemical  com- 
position remain  practically  unchanged. 

FRUITS  AND  VEGETABLES. — These  foods  are  for  the  most  part 
adapted  to  preservation  for  short  periods  at  ordinary  temperatures, 
and  cold  storage  at  a  temperature  slightly  above  zero  is  very  effective 
in  diminishing  the  rate  of  change  in  them.  The  humidity  of  the  storage 
chamber  should  be  kept  constant  at  about  60  per  cent  saturation  in 
order  to  diminish  evaporation  as  far  as  possible  without  favoring  the 
development  of  molds.  These  foods  generally  remain  alive  during 
storage  and  the  changes  due  to  intrinsic  enzymes  are  often  important. 
Some  fruits  need  to  undergo  further  -ripening  in  storage  before  they  are 
ready  for  consumption  and  this  change  may  be  accelerated  or  delayed 
by  changing  the  temperature  of  the  storage  chamber.  The  develop- 
ment of  bacteria  and  molds  with  consequent  rotting  is  best  delayed  by 
maintaining  dry  clean  fruits  and  vegetables  in  an  atmosphere  of  very 
constant  humidity  and  very  constant  temperature  slightly  above  the 
freezing-point. 

*  See  chapter  on  the  microbiology  of  butter. 


THE  PRESERVATION  OF  FOOD  BY  COLD          549 

LEGAL  CONTROL  OF  THE  COLD-STORAGE  INDUSTRY 

There  has,  been  a  rather  widespread  prejudice  against  cold-storage 
food  products,  and  in  some  respects  this  is  not  without  justification. 
Cold  storage  preserves  so  well  the  external  appearance  of  fresh  foods 
that  deception  in  the  sale  of  them  to  the  consumer  has  been  too  fre- 
quently practised.  This  is  extremely  unfortunate  for  all  parties 
concerned  in  such  transactions.  The  proper  branding  of  all  cold- 
storage  foods,  clearly  indicating  their  character  and  the  length  of  time 
held  in  storage,  would  ultimately  benefit  the  producer,  the  consumer 
and  also  the  cold-storage  industry.  Where  cold  storage  is  efficient  such 
a  practice  would  proclaim  its  efficiency.  Where  it  is  inefficient  the 
cold-storage  industry  can  ill  afford  to  allow  the  consumer  to  be  deceived 
concerning  the  food  he  is  purchasing.  The  strict  enforcement  of  laws 
compelling  the  proper  labeling  of  such  foods  and  prohibiting  their  sale 
except  when  branded  as  such  would  quickly  remove  unjust  prejudice 
against  cold  storage,  and  would  place  this  industry  upon  a  secure 
foundation,  greatly  increasing  the  possibilities  of  its  service  to  the  food 
producers  and  consumers,  and  at  the  same  time  promoting  the  legiti- 
mate interests  of  the  cold-storage  industry. 


CHAPTER  IV* 
THE  PRESERVATION  OF  FOOD  BY  CHEMICALS 

The  addition  of  preservative  substances  to  foods  is  a  very  ancient 
practice,  and  as  no  extensive  equipment  is  required  it  is  one  of  the 
cheapest  ways  of  preserving  food,  especially  on  a  small  scale.  The 
resulting  alteration  of  the  food  in  appearance  and  composition  is  greater 
than  when  it  is  preserved  by  cold  storage,  for  the  preservative  substance 
added  becomes  a  more  or  less  permanent  constituent  of  the  food,  but 
the  changes  are  not  necessarily  undesirable.  The  addition  of  chemical 
preservatives  is  often  practised  in  conjunction  with  desiccation  or  cold, 
or  solnetimes  even  in  canned  or  bottled  foods  sterilized  by  heat.  All 
the  substances  employed  as  preservatives  owe  whatever  efficiency  they 
may  possess  to  their  ability  to  restrict  the  activity  of  microorganisms, 
that  is,  their  antiseptic  properties. 

THE  EFFECTS  OF  PRESERVATIVES  UPON  FOODS  IN  GENERAL 

In  only  a  few  instances  are  chemical  preservatives  added  to  foods  to 
be  sold  as  fresh  foods,  and  these  practices  are  generally  regarded  with 
disfavor.  Their  most  important  use  is  in  the  prepared  foods,  the  pre- 
servative being  incorporated  with  the  food  during  the  process  of 
preparation  for  storage. 

THE  PROCESS  OF  CURING. — The  procedures  employed  necessarily 
vary  with  different  foods.  Physical  alterations  in  the  food,  such  as 
changes  in  form,  texture  and  water  content  are  usually  involved,  as 
well  as  the  solution  of  the  preservative  in  the  juices  of  the  food. 
Chemical  changes  due  to  the  intrinsic  enzymes  of  the  food,  to  the 
various  accessory  procedures  such  as  drying,  cooking  or  soaking  in 
pickling  solution  may  produce  marked  alteration.  In  some  cases  the 
preservative  reacts  chemically  with  some  constituent  of  the  food. 
During  the  curing  process  microbic  activity  may  be  more  or  less 
prominent  at  various  times,  playing  its  part  in  the  chemical  changes. 

*  Prepared  by  W.  J.  MacNeal. 

550 


THE  PRESERVATION  OF  FOOD  BY  CHEMICALS       551 

Bacteria,  yeasts  and  molds  are  likely  to  be  introduced  into  the  food  by 
the  various  manipulations  and  some  of  these  may  find  conditions  favor- 
able for  their  proliferation.  In  some  instances  the  activity  of  certain 
kinds  of  microbes  appears  to  be  essential  to  the  proper  curing  and 
subsequent  adequate  preservation  of  the  food;  the  preservative,  the 
constituents  of  the  food  and  the  microorganisms  mutually  reacting  to 
bring  about  the  desired  result.  It  is  worth  noting  that  the  added 
chemical  preservative  is  never  sufficiently  potent  to  destroy  with 
certainty  pathogenic  microbes  which  may  be  present  in  the  food. 

THE  PERIOD  OF  STORAGE. — Unless  the  food  has  been  sterilized  and 
stored  in  sealed  containers,  slow  changes  in  water  content,  in  physical 
appearance  and  in  chemical  composition  usually  take  place  during 
storage.  The  added  preservative  may  continue  to  react  with  the  food 
substance  and  its  decomposition  products.  During  this  period  there 
is  relatively  little  intimate  manipulation  of  the  food  and  therefore  little 
opportunity  for  the  penetration  of  new  microbes.  Some  of  those 
already  present  may  continue  their  activities  at  a  diminished  rate, 
producing  slow  chemical  changes  often  of  a  desirable  nature  rather  than 
otherwise.  Accessory  conditions,  such  as  desiccation,  cold  storage, 
or  sterilization  and  sealing,  may  greatly  retard  or  check  altogether 
microbic  activity. 

THE  AFTER-STORAGE  CHANGES. — The  immediate  preparation  of 
preserved  food  for  consumption  is  frequently  important.  The  pre- 
servative may  be  largely  removed  mechanically,  or  extracted  with 
water.  During  cooking  peculiar  chemical  reactions  may  occur,  and 
cooking  is  also  important  in  the  destruction  of  microorganisms  remain- 
ing alive  in  the  food  up  to  that  time. 

THE  CHEMICAL  PRESERVATION  OF  CERTAIN  FOODS 

MEATS  AND  FISH. — The  preservation  of  meat  and  of  fish  by  salting 
depends  largely  upon  the  increase  of  osmotic  tension  in  the  food,  a 
physical  change  sufficient  to  prevent  or  greatly  delay  the  growth  of 
microorganisms.  Sodium  chloride  (NaCl)  probably  owes  its  preserva- 
livi-  value  solely  to  this  physical  effect.  In  practice  its  action  is  often 
supplemented  by  the  addition  of  a  small  amount  of  saltpeter  (KNO3), 
and  sometimes  also  cane  sugar  (Ci2H22Oii).  The  fluids  of  the  flesh 
are  in  part  removed  by  this  treatment,  carrying  away  a  part  of  the 
soluble  constituents.  The  fluids  which  remain  contain  the  added 


552  MICROBIOLOGY    OF   FOODS 

preservative  substance  in  solution,  and  the  whole  mass  of  food  sub- 
stance is  permeated  by  them.  Potassium  nitrate  (saltpeter)  reacts 
with  the  flesh,  being  reduced  in  part  to  nitrite.  This  enters  into  a 
combination  with  the  coloring  matter  of  meat,  which  upon  cooking 
produces  the  characteristic  red  color  of  meat  cured  with  saltpeter. 

The  various  manipulations  during  the  process  of  pickling  or  dry 
curing  serve  to  introduce  numerous  microorganisms.  Many  of  these 
may  flourish  in  the  pickling  fluids,  but  in  a  sufficient  concentration  of 
salt  and  at  a  sufficiently  low  temperature,  decomposition  ordinarily  does 
not  progress  so  as  to  become  objectionable,  and  proteolytic  decom- 
position (putrefaction)  is  effectually  prevented.  This  protection  of 
the  protein  depends  to  some  extent  upon  the  acidity  of  the  medium, 
which  in  turn  is  due  largely  to  the  bacterial  decomposition  of  the 
muscle  sugar.  The  powerful  putrefactive  bacteria  (B.  aedematis 
group)  flourish  only  in  an  alkaline  medium.  On  the  other  hand,  too 
high  a  degree  of  acidity  becomes  in  itself  objectionable  on  account  of 
the  sour  or  rancid  taste,  and  it  is,  therefore,  important  that  the  acid- 
producing  bacteria  should  be  held  in  check  somewhat.  In  practice, 
saltpeter  has  proved  of  value  for  this  particular  purpose,  and  its 
action  apparently  depends  upon  the  antiseptic  effect  of  minute  quan- 
tities of  nitric  acid  (HNOs)  and  nitrous  acid  (HNO2)  set  free  from  the 
salt  by  the  excess  of  organic  acids  produced  by  the  bacteria.  The 
curing  of  meats  by  pickling  solutions  is  often  supplemented  by  desicca- 
tion and  impregnation  with  the  antiseptic  substances  of  wood  smoke. 

The  dry-salting  of  codfish  is  an  example  of  preservation  by  increasing  the  osmotic 
tension.  The  fish  is  cleaned  and  beheaded,  split  longitudinally,  and  the  vertebral 
column  removed.  It  is  then  carefully  washed,  and  all  visible  blood  is  removed.  The 
pieces  are  next  covered  with  dry  salt  and  packed  in  open  casks.  The  salt  rapidly 
extracts  water  from  the  flesh  and  a  strong  brine  results.  After  a  few  days  the  casks 
are  emptied  out,  and  the  pieces  of  fish,  now  smaller  because  of  the  loss  of  water,  are 
again  thoroughly  washed  and  again  packed  in  dry  salt  so  that  the  adjacent  pieces  of 
fish  are  completely  separated  by  an  intervening  layer  of  solid  salt.  The  contents 
of  the  cask  are  subjected  to  high  pressure  to  remove  air,  and  the  cask  is  finally  closed. 

The  curing  of  ham  is  an  example  of  preservation  by  increased  osmotic  tension 
combined  with  the  addition  of  chemical  preservatives.  After  slaughter  and  chilling, 
the  hams  are  injected  with  a  solution  containing  25  per  cent  common  salt,  15  percent 
granulated  sugar,  and  12  per  cent  saltpeter,  and  are  then  stored  at  a  low  tem- 
perature, preferably  between  o°  and  +4°,  in  a  brine  containing  about  20  per  cent 
common  salt,  5  per  cent  sugar,  and  i  per  cent  saltpeter.  The  brine  is  renewed  once 
or  twice  at  intervals  of  a  week  or  ten  days.  After  about  a  month  the  hams  are 


THE    PRESERVATION    OF    FOOD   BY    CHEMICALS  553 

washed  in  warm  water,  dried  and  hung  in  wood  smoke  for  several  days.  They  are 
then  stored  in  a  cool  place.  The  proportions  of  the  various  constituents  of  the 
pickling  solutions  are  subject  to  rather  wide  variation,  and  in  general,  it  may  be 
said  that  the  higher  the  temperature  of  the  storage  room,  the  more  concentrated 
must  be  the  pickling  solutions  to  insure  satisfactory  preservation. 

DAIRY  PRODUCTS. — Butter  is  usually  salted  with  sodium  chloride 
to  impart  the  desired  taste,  and  this  salt  also  acts  to  some  extent  as  a 
preservative  by  increasing  the  osmotic  tension  of  the  moisture  remain- 
ing in  the  butter.  Antiseptics  such  as  boric  acid,  saltpeter,  salicylic 
acid  and  formaldehyde  have  been  employed  in  the  preservation  of 
butter,  the  first- mentioned  appearing  to  be  the  most  satisfactory. 
One  half  of  i  per  cent  of  boric  acid  incorporated  with  high-grade  butter 
previous  to  storage  greatly  delays  rancid  change. 

.Fresh  milk  and  cream  are  also  sometimes  treated  with  antiseptics 
such  as  formaldehyde,  but  the  use  of  any  chemical  preservative  what- 
ever in  these  dairy  products  is  unnecessary  and  generally  disapproved. 

PREPARED  VEGETABLE  AND  FRUIT  FOODS. — These  foods  are  some- 
times preserved  by  vinegar,  sugar  or  alcohol,  the  presence  of  which  is  of 
course  very  evident  to  the  consumer.  Other  substances  less  readily 
detected,  such  as  sulphurous  acid  and  sulphites,  boric  acid,  salicylic  acid, 
benzoic  acid  and  sodium  benzoate,  and  formaldehyde,  are  also  em- 
ployed in  foods  which  must  be  kept  some  time  after  exposure  to  the  air. 
These  substances  are  incorporated  with  the  food  before  it  is  packed, 
and  serve  to  inhibit  the  activity  of  microorganisms  which  gain  access 
to  it. 

THE  NUTRITIVE  VALUE  OF  PRESERVED  FOODS 

The  nutritive  value  of  a  food  depends  upon  the  amount  of  utilizable 
food  principles  it  contains.  The  food-principle  content  can  be  readily 
measured  by  chemical  analysis,  and  in  general  there  is  no  important 
difference  between  a  preserved  food  and  the  corresponding  fresh  food 
in  this  respect.  The  utilization  of  the  food  principles,  however, 
depends  upon  a  number  of  factors  and  may  be  greatly  influenced  by 
individual  peculiarities  of  the  consumer.  One  important  factor  in  the 
utilization  of  a  food,  and  probably  the  most  important  factor  in  deter- 
mining its  market  value,  is  palatability.  In  general,  preserved  foods 
are  pleasant  to  the  taste  when  eaten  at  intervals,  but  upon  long  con- 
tinued daily  ingestion,  the  appetite  for  them  fails  and  they  may  even 


554  MICROBIOLOGY   OF   FOODS 

become  distasteful.  It  would,  therefore,  appear  to  be  erroneous  to 
regard  preserved  foods  as  in  every  respect  as  valuable  from  the  stand- 
point of  nutrition  as  the  corresponding  fresh  foods.  The  difference  is 
not  dependent  upon  a  change  in  the  food-principle  content,  but  must  be 
sought  rather  in  slightly  altered  composition  of  the  food  and  the 
specific  effects  of  newly  formed  substances,  and  especially  in  the 
possible  effects  of  the  continued  ingestion  of  the  contained  chemical 
preservatives  upon  the  consumer. 

THE  EFFECTS  OF  FOOD  PRESERVATIVES 

The  essential  characters  of  a  food  preservative  include  antiseptic 
action  to  prevent  decomposition  of  the  food,  and  absence  of  evident 
poisonous  or  deleterious  influence  upon  the  consumer.  It  follows  there- 
fore that  the  effects  of  food  preservatives  upon  the  consumer,  if  they 
exist  at  all,  are  at  any  rate  not  easily  recognized,  and  on  account  of  the 
economic  importance  of  the  questions  here  involved,  this  field  of 
scientific  research  has  been  energetically  cultivated  by  investigators 
with  different  viewpoints,  and  the  results  of  investigation  have  been 
discussed  with  some  heat.  The  passage  of  the  U.  S.  Food  and  Drugs 
Act  was  followed  by  considerable  discussion  of  these  questions.  Gradu- 
ally the  practical  administration  of  the  law  has  become  more  settled 
and  the  use  of  many  food  preservatives  is  still  permitted. 

SUBSTANCES  WHICH  PRESERVE  BY  THEIR  PHYSICAL  ACTION. — The 
preservative  effects  of  sodium  chloride  seem  to  depend  upon  the  high 
osmotic  tension  of  strong  salt  solution,  and  the  same  may  be  said 
of  cane  sugar.  When  diluted  so  as  to  be  eaten  with  relish,  these 
substances  are  themselves  properly  classed  as  foods,  without  deleterious 
effects  upon  ordinary  individuals. 

SUBSTANCES  WHICH  PRESERVE  BY  THEIR  CHEMICAL  ACTION. — 
These  preservatives  inhibit  the  activity  of  microorganisms  in  a  dif- 
ferent way,  not  by  withdrawing  water  from  the  microbic  cell,  but  by 
entering  into  chemical  combination  with  the  living  substance  in  such  a 
way  as  to  hinder  its  activity,  or  by  entering  into  chemical  reactions 
with  the  food  to  produce  new  substances  capable  of  attacking  the 
microbic  protoplasm  in  this  way.  The  ideal  chemical  food  preservative 
would  be  one  which,  without  altering  the  food  substance,  would  ex- 
hibit this  poisonous  property  toward  living  protoplasm  until  the  food 
was  ready  for  consumption,  and  then  would  suddenly  and  permanently 


THE  PRESERVATION  OF  FOOD  BY  CHEMICALS       555 

lose  this  property.     None  of  the  ordinary  food  preservatives  approaches 
this  ideal  very  closely. 

INORGANIC  FOOD  PRESERVATIVES. — Boric  acid  and  borax  are  weak 
antiseptics,  practically  a  saturated  solution  of  boric  acid  being  neces- 
sary to  inhibit  ordinary  bacterial  growth.  When  employed  as  a  dry 
wder  on  the  surface  of  meats,  boric  acid  prevents  the  growth  of  mold, 
d  most  of  it  is  removed  from  the  food  before  consumption.  When 
corpora  ted  with  butter  it  is  eaten,  and  0.5  to  i.o  g.  may  be  taken 
ily  in  this  way  alone.  The  effect  of  such  amounts  of  boric  acid  upon 
the  consumer  is  still  a  disputed  question.  Wiley,*  after  an  extensive 
investigation,  concluded  that  small  doses  of  either  boric  acid  or  borax 
continuously  administered  for  long  periods  create  disturbances  of 
health. 

Nitric  acid  and  nitrous  acid  and  their  salts  are  food  preservatives 
of  some  theoretical  interest  because  it  is  well  known  that  some  bacteria 
readily  decompose  fairly  strong  solutions  of  nitrates,  and  also  oxidize 
or  reduce  nitrites.  Apparently,  however,  this  is  true  only  in  neutral 
or  alkaline  solutions,  and  in  the  presence  of  free  acid  the  activity  of 
these  microbes  is  quickly  inhibited.  The  preservative  effect  of  nitrates 
and  nitrites  is  best  ascribed  to  the  liberation  of  minute  quantities  of 
free  nitric  and  nitrous  acids  from  these  salts,  and  these  substances  are 
without  value  as  preservatives  in  foods  which  are  alkaline  in  reaction. 
The  effect  of  the  ingestion  of  nitrate  or  foods  preserved  with  nitrate 
upon  the  consumer  has  been  investigated  by  Wiley,  who  concluded 
that  the  deleterious  effects  are  slight  and  less  clearly  detected  than  in 
the  case  of  the  other  preservatives.  Small  quantities  of  saltpeter 
appreciably  delay  the  digestive  action  of  a  mixture  of  pepsin  and 
hydrochloric  acid  (artificial  gastric  juice)  upon  coagulated  egg  white  in 
a  test  tube.f  Minute  but  variable  amounts  of  nitrites  occur  in  foods 
preserved  with  nitrates,  but  whether  these  amounts  are  sufficient  to 
produce  the  specific  nitrite  effect  upon  the  blood  circulation  of  the 
consumer  has  not  yet  been  definitely  ascertained. 

Sulphurous  acid  and  the  sulphites  are  rather  extensively  used  in 
chopped  meat  (Hamburg  steak)  and  in  cider  and  wines.  The  addition 
of  sulphite  to  chopped  meat  serves  a  three-fold  purpose,  retarding  bac- 
terial decomposition,  producing  a  red  color  on  the  exposed  surface,  and 

*  U.  S.  Dept.  Agr..  Bureau  of  Chemistry,  Bull.  No.  84,  Part  I. 

t  MacNeal  and  Kerr:  Unpublished  work. 


556  MICROBIOLOGY    OF   FOODS 

removing  odors  of  decomposition.  It  thus  not  only  delays  decomposi- 
tion, but  also  to  a  certain  extent  masks  the  decomposition  which  has 
already  occurred.  The  ingestion  of  moderate  quantities  of  sulphites 
in  food  has  at  times  been  followed  by  acute  gastric  derangement  in 
man,  and  prolonged  feeding  of  meat  containing  sulphites  has  been 
followed  by  inflammatory  changes  in  the  kidneys  of  experimental 
animals. 

Fluorides  have  been  used  to  a  slight  extent  in  beverages,  but  acute 
gastric  derangement  and  depression  of  the  heart  are  caused  by  rather 
small  quantities,  and  probably  on  this  account  the  salts  of  hydrofluoric 
acid  have  not  come  into  very  general  use  as  food  preservatives. 

ORGANIC  FOOD  PRESERVATIVES. — Formic  acid  (H-COOH)  and 
acetic  acid  (CHVCOOH)  are  produced  by  microbic  activity  and  their 
preservative  action  appears  to  depend  more  upon  the  degree  of  acidity 
than  upon  the  character  of  the  acid  radical.  Both  these  acids  appear 
to  be  utilized  as  food  in  the  body  of  the  consumer. 

Benzoic  acid  and  benzoates  are  rather  extensively  employed  in 
prepared  vegetable  food  products,  such  as  jams  and  catsups.  The 
antiseptic  effect  seems  to  be  due  wholly  to  free  benzoic  acid,  even  where 
it  is  added  in  the  form  of  the  salt,  but  the  action  is  not  due  merely  to 
the  acidity  (i.e.,  the  hydrogen  ion).  Benzoic  acid  is  not  utilized  as  a 
food  in  the  body,  but  is  excreted  by  the  .kidneys  in  the  form  of  hippuric 
acid.  It  has  been  said  to  produce  irritant  effects  upon  the  stomach 
and  the  kidneys,  and  to  arrest  the  action  of  digestive  enzymes  in  dilute 
solutions,  but  the  Referee  Board*  of  the  United  States  Department  of 
Agriculture,  after  extensive  investigations,  concluded  that  small  doses 
of  sodium  benzoate  mixed  with  food  are  not  injurious  to  health,  and 
do  not  impair  the  quality  or  nutritive  value  of  the  food. 

Salicylic  acid  and  the  salicylates  have  been  used  for  much  the  same 
purposes  as  benzoic  acid,  and  there  does  not  appear  to  be  much  dif- 
ference between  the  two  acids,  either  in  their  efficiency  as  preservatives 
or  in  their  possible  deleterious  effects  upon  the  consumer.  Salicylic 
acid  is  more  expensive.  After  extensive  investigation  Wiley f  has 
concluded  that  the  addition  of  salicylic  acid  and  salicylates  to  foods  is 
reprehensible  in  every  respect,  this  conclusion  corresponding  to  the 
results  of  similar  work  by  the  same  investigator!  upon  benzoic  acid. 

•  U.  S.  Dept.  Agr.  Report  No.  88,  May,  1909. 

t  U.  S.  Dept.  Agr.,  Bureau  of  Chemistry,  Bull.  No.  84,  Part  II,  1906. 

J  U.  S.  Dept.  Agr.,  Bureau  of  Chemistry,  Bull.  No.  84.  Part  IV,  1908. 


THE  PRESERVATION  OF  FOOD  BY  CHEMICALS       557 

Formaldehyde  is  very  efficient  as  an  antiseptic,  delaying  microbial 
decomposition  when  added  to  foods  in  very  small  quantity.  Its  use 
for  this  purpose  is  generally  condemned,  partly  because  of  its  hardening 
or  "fixing"  effect  upon  the  protein  constituents  of  the  food,  tending  to 
make  them  more  indigestible.  Its  use  in  milk  and  milk  products, 
though  still  practised  to  some  extent,  has  been  prohibited  by  law  in 
some  states. 

Alcohol  (CH3-CH2OH),  in  sufficient  concentration,  is  an  excellent 
preservative,  but  its  presence  in  foods  is  readily  detected,  and  it  gives 
rise  to  characteristic  effects  upon  the  consumer.  Furthermore,  such 
foods  are  subject  to  special  taxation  as  alcoholic  products.  Its  use 
as  a  food  preservative  is  therefore  limited. 

Wood  smoke  has  been  employed  for  centuries  in  the  curing  of  meats. 
Its  antiseptic  properties  probably  depend  for  the  most  part  upon 
creosote  and  pyroligneous  acid,  constituents  of  wood  smoke  which  are 
antiseptic  and  also  undoubtedly  poisonous  in  sufficient  doses.  Smok- 
ing is  a  time-honored  custom,  however,  and  the  amount  of  these 
substances  consumed  with  the  smoked  meat  is  doubtless  exceedingly 
minute. 

SUBSTANCES  ADDED  TO  FOODS  TO  IMPROVE  THE  APPARENT  QUALITY. 
Several  chemical  substances  are  employed  in  various  foods  to  im- 
prove the  appearance,  or  to  simulate  the  taste  of  a  higher-grade  product. 
In  some  cases  the  presence  of  these  agents  is  known  to  the  consumer, 
and  desired  by  him;  in  other  instances  they  are  employed  to  deceive 
the  purchaser.  Butter  coloring  is  quite  generally  used  to  produce  the 
color  of  June  butter  the  year  around;  nitrates  bring  about  a  pleasing 
red  color  in  cooked  pickled  meats;  copper  sulphate  is  used  to  give  a 
more  brilliant  green  color  to  prepared  vegetable  foods;  sulphites  restore 
the  red  color  of  freshly  cut  meat  to  meat  far  from  fresh;  saccharine 
devoid  of  food  value  gives  a  taste  resembling  sugar  to  a  variety  of 
preparations  at  a  great  saving  in  cost  to  the  manufacturer;  carbonates 
of  the  alkalies  or  alkaline  earths,  added  to  milk,  neutralize  the  acids 
resulting  from  bacterial  decomposition  and  so  keep  the  milk  sweet; 
inorganic  acids  added  to  weak  vinegar  increase  its  acidity.  Some  of 
these  practices  are  so  universal  and  so  well  known  that  they  are  no 
longer  criticized;  others,  such  as  the  use  of  chalk  in  milk,  are  generally 
disapproved. 


558  MICROBIOLOGY   OF- FOODS 

LEGAL  CONTROL  OF  THE  PRESERVATION   OF  FOODS  BY   CHEMICALS 

The  desirability  of  legal  regulation  of  the  use  of  chemical  food  pre- 
servatives  is  now  generally  recognized,  but  there  is  still  considerable 
diversity  of  opinion  concerning  what  this  regulation  should  be.  Few, 
indeed,  maintain  that  a  substance  exerting  antiseptic  action  upon 
microorganisms  outside  the  body  is  wholly  without  influence,  after 
i  ngestion,  upon  the  enzymes  and  bacteria  of  the  normal  digestive  tract, 
even  if  we  disregard  the  possible  effects  of  the  substance  after  absorp- 
tion. It  seems  necessary  to  grant  the  existence  of  some  effect,  even 
though  it  be  so  slight  as  to  have  escaped  detection.  Over  against  the 
possible  injury  to  the  consumer  must  be  placed  the  economic  saving 
through  the  use  of  the  preservative,  often  involving  a  considerable 
amount  of  money.  In  the  absence  of  accurate  and  trustworthy 
knowledge  concerning  the  actual  influence  of  preservatives  in  the  human 
body  it  would  seem  wise  to  prohibit  all  deception  in  regard  to  their 
presence.  The  principle  advocated  by  Pasteur  (1891)  would  still  seem 
to  be  best,  that  is,  to  allow  the  use  of  preservatives,  which  are  not 
known  to  be  dangerous,  upon  the  condition  that  their  presence  and  the 
exact  amounts  be  definitely  and  clearly  stated  on  an  appropriate  label 
for  the  benefit  of  the  purchaser  and  the  ultimate  consumer.  Such 
regulation  would  not  only  protect  the  consumer  against  deception  and 
fraud  but  would  go  far  toward  removing  prejudice  against  preservatives, 
for  even  now  there  is  little  or  no  objection  to  those  preservative  sub- 
stances of  which  the  presence  and  the  amount  can  be  detected  and 
roughly  measured  by  the  senses,  such  as  salt,  sugar,  spices,  vinegar 
and  wood  smoke. 


CHAPTER  V 
MICROBIOLOGY  OF  FERMENTED  FOODS 

COMPRESSED  YEAST* 

It  is  stated  that  bread  over  four  thousand  years  old  from  the  tombs 
of  ancient  Egypt  has  been  found  to  contain  dead  yeast  cells,  indicating 
the  antiquity  of  the  use  of  yeast  in  bread  making.  The  leavened 
bread  of  the  ancients  doubtless  contained  yeast  in  combination  with 
other  microorganisms.  " Potato  yeast  starters"  for  bread  making 
were  a  later  development  but  antedate  the  manufacture  of  compressed 
yeast  as  we  now  know  this  industry.  With  the  development  of  brew- 
ing it  was  found  that  the  yeast  left  in  the  fermentation  vats  was  suitable 
for  bread  making.  In  England  a  great  deal  of  brewers'  yeast  is  still 
used  for  this  purpose;  housewives,  rather  than  commercial  bakers,  are 
the  principal  users  of  this  yeast.  Experience  has  shown  that  brewers' 
yeast  is  weaker  than  distillers'  yeast  and,  therefore,  less  suitable  for 
breadmaking;  brewers'  yeast  also  is  often  very  bitter  from  the  presence 
of  hop  resins.  Because  of  these  facts  commercial  bakers  came  to 
prefer  distillers'  yeast,  which  at  first  was  a  by-product  of  the  alcohol 
or  potato  spirits  factory.  This  by-product  yeast  varied  greatly  in 
strength  and  general  quality  and  a  demand  arose  for  a  yeast  of  more 
uniform  character.  As  a  result  of  this  demand  suitable  pure  strains  of 
yeast  were  selected  and  propagated  for  breadmaking — the  alcohol  in 
this  case  itself  becoming  a  by-product  and  yeast  the  primary  product. 
The  compressed  yeast  and  yeast  cake  industry  has  now  reached  great 
proportions  and  represents  a  very  striking  example  of  the  successful 
application  of  science  to  industry. 

Although  the  manufacturing  processes  are  carefully  controlled 
analytically  and  bacteriologically  and  although  a  great  deal  of  valuable 
research  work  has  been  done  in  recent  years,  little  of  the  information 
thus  obtained  has  been  made  available  to  microbiologists.  However, 
the  Wahl-Henius  Baking  Institute  and  others  are  doing  much  to  dis- 
seminate and  to  increase  the  existing  knowledge  on  the  subject. 

*  Prepared  by  W.  V.  Cruess. 

559 


560  MICROBIOLOGY   OF.  FOODS 

The  manufacture  of  compressed  yeast  begins  with  the  preparation  of  the  malt. 
Two  cereals,  barley  and  rye,  are  commonly  used  in  most  factories — some  use  barley 
only.  The  barley  is  first  "steeped"  in  cold  water  as  for  brewing  (page  623).  It 
is  then  placed  in  large  slowly  revolving  drums  maintained  by  a  current  of  moist  cool 
air  at  10°  to  25°.  The  air  is  cooled  by  being  drawn  through  water  sprays  and  wet 
coke.  The  usual  temperature  is  about  15°  to  18°.  The  barley  is  allowed  to 
germinate  until  the  acrospire  (embryo  blade)  has  reached  the  length  of  the  kernel. 
Rootlets  several  times  the  length  of  the  kernel  form  and  cause  the  sprouted  barley 
to  mat  together. 

The  object  of  malting  is  to  cause  the  barley  through  the  process  of  germination 
to  form  large  amounts  of  diastase  and  of  proteases.  The  formation  of  the  latter 
enzymes  is  as  important  as  that  of  diastase  because  the  yield  and  vigor  of  the  yeast 
depend  upon  the  amount  of  assimilable  nitrogen  compounds  present  in  the  nutrient 
liquid.  These  are  derived  largely  from  the  complex  insoluble  proteins  of  the  grain 
by  the  hydrolyzing  action  of  protein- splitting  enzymes.  Too  short  a  period  of  too 
high  a  temperature  of  malting  results  in  low  yields  of  the  enzymes;  too  long  a 
period  results  in  loss  of  enzymes  formed  during  germination  and  loss  of  starch. 
Under  normal  conditions  of  temperature  and  humidity  about  five  to  seven  days' 
malting  is  used  for  barley.  A  typical  record  of  temperature  and  rate  of  rotation  of 
a  drum  during  malting  is  given  by  Wahl-Henius  as  follows: 

Day  Temperature  Period  of  Rotation 

ist  13°  Once  in  2  hours. 

2d  1 6°  Once  in  2  hours. 

3d  1 8°  Once  in  2  hours. 

4th  21°  Once  in  i  ^  hours. 

5th  24°  Once  in  40  minutes. 

Rye  is  said  to  be  used  to  furnish  protein  and  mineral  salts  which  increase  the  yield 
and  vigor  of  the  yeast.  It  is  malted  on  cement  floors  in  layers  about  eight  inches 
thick.  Much  less  of  the  rye  is  used  than  of  the  barley.  The  sprouted  barley  and 
rye  grains  are  crushed  between  rough  steel  rolls  which  revolve  at  different  speeds, 
the  difference  in  speed  resulting  in  a  grinding  action  upon  the  grains.  The  malt  is 
not  dried  before  grinding;  in  this  respect  the  practice  is  different  from  that  fol- 
lowed in  brewing.  Drying  results  in  loss  of  enzyme-content  and  increases  the  cost 
of  operation  without  in  any  way  improving  the  quality  of  the  yeast. 

The  crushed  malt  is  mixed  with  water  in  a  large  wooden  tank  known  as  the 
"Mash  tun"  and  which  is  equipped  with  revolving  stirring  arms  and  steam  coils. 
The  coils  are  usually  controlled  by  a  thermostat  in  order  that  an  exact  temperature 
may  be  maintained  in  the  mash  tun  during  acidification  of  the  mash. 

In  the  mash  tun  the  starch  is  converted  into  maltose;  a  high  concentration  of 
lactic  acid  is  formed  and  the  insoluble  proteins  of  the  grains  are  converted  to  a  con- 
siderable degree  into  peptones  and  to  a  certain  extent  into  amino  acids.  The  forma- 
tion of  acid  and  hydrolysis  of  the  proteins  are  obtained  largely  by  maintaining  a 
temperature  of  50°  to  52°.  A  vigorous  culture  of  a  lactic  acid  forming  organism  is 
added  to  the  mash  as  a  starter.  B.  delbruckii  is  often  used  for  the  purpose — in 


MICROBIOLOGY   OF   FERMENTED    FOODS  561 

some  cases  natural  cultures  are  used.  The  pure  cultures  of  selected  strains  give  the 
best  results.  These  are  grown  at  50°  to  52°  in  small  thermostat  regulated  tanks. 
At  temperatures  much  below  50°  butyric  acid-forming  organisms  will  develop;  if 
the  temperature  is  much  higher  than  this,  the  lactic  organisms  are  weakened  and 
peptonization  of  the  proteins  is  halted  by  destruction  of  proteases  and  coagulation 
of  the  barley  and  rye  proteins.  Diastase  works  more  rapidly  at  temperatures  of 
60°  to  65°  than  at  50°  but  gives  a  much  higher  proportion  of  maltose  to  dextrins  at 
the  lower  temperature. 

Lactic  acid  activates  the  proteases  and  checks  or  prevents  the  growth  of  undesir- 
able bacteria  such  as  B.  subtilis  during  yeast  growth  or  storage  of  the  finished  yeast. 
Success  or  failure  depends  as  much  upon  the  proper  acidification  of  the  mash  as 
upon  any  other  single  factor. 

Diastase  rapidly  converts  the  starch  to  maltose  but  peptonization  of  the  pro- 
teins is  an  extremely  slow  process.  Where  three  to  four  hours  would  suffice  for  the 
diastase  eighteen  to  thirty  should  be  allowed  to  permit  the  proteases  to  act.  The 
yield  of  yeast  depends  upon  the  degree  of  protein  hydrolysis.  This  long  period  of 
mashing  can  only  be  carried  out  by  use  of  lactic  acid  cultures  and  accurate  regulation 
of  the  temperature  to  favor  the  growth  of  the  lactic  organisms  and  to  check  the 
growth  of  other  types. 

During  mashing  the  plastic  mass  of  crushed  malt  and  water  becomes  thin  and 
watery  because  of  conversion  of  starch  to  sugar.  This  change  permits  the  liquid  to 
be  drained  from  the  grain  husks  and  the  husks  to  be  washed  with  water.  The 
liquids  so  obtained  are  combined  and  filtered.  The  filtered  liquid  is  of  n  to  14 
degrees  Balling  and  acid  in  flavor.  In  some  factories  the  mash  before  straining  or 
the  liquid  after  straining  is  heated  to  a  temperature  sufficient  to  destroy  the  lactic 
organisms;  in  others  this  pasteurization  is  omitted. 

The  liquid  now  known  as  "wort"  is  cooled  to  the  pitching  (yeast  inoculation) 
temperature  of  about  20°  to  25°.  The  cooled  liquid  is  transferred  to  large  open  vats, 
usually  of  10,000  to  15,000  gallons  capacity,  either  of  wood  or  of  enameled  or  otherwise 
protected  steel.  §  These  are  equipped  with  large  copper  cooling  coils  through  which 
water  may  be  circulated  to  prevent  too  great  a  rise  in  temperature  of  the  wort  from 
the  heat  of  fermentation.  Too  high  temperatures  favor  alcoholic  fermentation  with 
low  yields  of  yeast.  Compressed  air  is  delivered  to  the  liquid  from  perforated  coils 
placed  in  the  bottom  of  the  vat.  Vigorous  aeration  is  used  to  favor  the  production 
of  yeast  and  to  limit  alcohol  formation. 

Pure  cultures  of  selected  strains  of  yeast  are  employed  for  "pitching"  purposes. 
This  yeast  starter  is  grown  in  sterilized  wort  in  a  small  vat  which  can  be  kept  free 
from  undesirable  organisms.  This  pure  culture  is  replaced  frequently  by  new  start- 
ers propagated  from  pure  cultures  made  by  standard  plating  methods.  This 
practice  is  necessary  to  insure  purity  of  the  yeast  turned  out  by  the  large  vats. 
Care  must  be  taken  to  insure  that  the  yeast  used  is  true  to  type. 

Fermentation  and  yeast  growth  in  the  large  vats  proceed  for  about  fourteen  to 
twenty-four  hours.  In  some  factories,  the  yeast  is  then  allowed  to  settle.  This 
takes  place  rapidly  and  almost  completely  if  an  agglomerating  (granule-forming) 
type  of  yeast  is  used,  very  slowly  if  the  yeast  is  of  the  fine-grained  type.  If  settling 


562  MICROBIOLOGY   OF   FOODS 

can  be  accomplished,  later  operations  are  simplified,  but  settling  is  not  essential  to 
success. 

The  fermented  liquid  and  yeast  from  the  vats  are  passed  through  continuous 
centrifuges  similar  in  design  to  milk  centrifuges.  A  creamy  suspension  of  yeast 
and  liquid  is  obtained  from  one  outlet  and  a  liquid  almost  yeast-free  from  the 
other  outlet  of  the  centrifuge. 

The  creamy  suspension  of  yeast  is  chilled  by  flowing  over  brine-cooled  coils  and 
flows  to  the  filter  supply  tank.  Chilling  the  yeast  checks  the  growth  of  slime-form- 
ing and  other  harmful  organisms. 

The  mixture  of  yeast  and  liquid  is  filter-pressed  to  remove  excess  of  liquid.  The 
pasty  mass  of  yeast  forming  the  filter  press-cakes  is  usually  mixed  with  a  small 
amount  of  starch  which  gives  a  friable  texture  to  the  yeast.  A  small  amount  of 
vegetable  oil  is  used  in  other  plants  for  the  same  purpose.  The  yeast  is  next  molded 
and  cut  into  blocks  of  the  desired  shape  and  size.  After  wrapping  it  is  held  in  cold 
storage  until  sold.  At  room  temperatures  the  yeast  liquefies  through  autolysis  or 
becomes  slimy  from  bacterial  growth,  moldy  or  weakened.  Therefore,  keeping  the 
yeast  at  a  low  temperature  is  essential  to  longevity  of  the  product. 

The  waste  liquor  from  the  centrifuges  and  filter  press  is  distilled  in  a  continuous 
still.  The  alcoholic  distillate  is  diluted  to  about  10  per  cent,  alcohol  and  passed 
through  charcoal  or  coke  filled  generators  to  produce  distilled  vinegar  used  prin- 
cipally by  pickle  manufacturers.  The  vinegar  is  sometimes  used  for  aging,  or,  di- 
luted to  5  per  cent  acetic  acid,  is  sold  for  domestic  use  (See  Division  VI,  Chapter  IV). 

Compressed  yeast  is  also  mixed  with  corn  meal  and  compressed  into  cakes  and 
dried  at  temperatures  low  enough  not  to  affect  seriously  the  yeast's  vitality.  The 
dried  product  is  known  and  used  in  rural  communities  as  "Magic  Yeast,"  etc. 
This  product  makes  it  possible  to  obtain  relatively  pure  and  active  yeast  in  isolated 
communities.  Such  yeast  gives  the  best  results  if  made  into  a  thin  batter  or  potato 
yeast-starter  twenty-four  hours  before  it  is  to  be  used  in  bread  making. 

The  manufacture  of  compressed  yeast  is  carefully  controlled  by  chemical  analysis 
and  frequent  microscopical  examinations.  Good  compressed  yeast  should  be  slightly 
moist  but  not  "sloppy;"  the  color  should  be  creamy  white,  and  it  should  show  a 
fine  fracture  when  broken.  It  should  melt  readily  on  the  tongue.  The  flavor  should 
be  clean  and  free  from  any  suggestion  of  butyric  acid  or  putrefaction.  It  should 
show  only  a  very  few  dead  cells  when  the  cells  are  mounted  in  a  dilute  solution  of 
methylene  blue.  It  will  normally  contain  a  few  lactic  bacteria  but  must  be  free 
from  B.  subtilis  or  putrefactive  organisms.  In  a  case  that  came  to  the  writer's 
attention  the  yeast  from  a  certain  factory  carried  rather  large  numbers  of  B.  subtilis 
which  caused  the  yeast  to  become  slimy.  The  infection  was  traced  to  the  water  used 
in  the  factory.  In  another  case  the  yeast  was  found  to  be  the  cause  of  ropy  bread 
because  of  its  contamination  with  B.  mesentericus  mdgatus.  The  use  of  a  mash 
highly  acidified  by  growth  of  B.  delbruckii  or  other  lactic  organisms  will  hold  in 
check  most  "yeast  disease"  organisms. 


MICROBIOLOGY    OF    FERMENTFD    FOODS  563 

Yeast  as  Food. — Yeast  is  very  rich  in  protein  and  forms  a  readily 
assimilable  human  food  or  stock  food.  Breweries  and  distilleries 
produce  very  large  quantities  of  yeast  as  a  by-product.  In  the  case  of 
breweries,  the  yeast  carries  a  considerable  quantity  of  hop  resins  which 
impart  a  bitter  taste  to  the  product  and  render  it  unpalatable  for  human 
food.  It  is,  however,  fed  successfully  to  stock  with  the  spent  grains. 
By  suitable  washing  processes  most  of  the  bitter  resins  may  be  removed. 

In  addition  to  its  nutritive  value  it  is  claimed  by  some  that  yeast 
taken  internally  or  applied  externally  possesses  remarkable  healing 
properties  for  wounds,  boils,  pimples,  etc.  It  is  also  claimed  that  yeast 
is  rich  in  vitamins,  the  now  popular  growth-producing  compounds. 
These  contentions  are,  however,  not  yet  fully  substantiated  by  thorough 
investigations. 

Yeast  may  be  dried  successfully  at  moderate  temperatures,  but 
tends  to  caramelize  or  darken  at  temperatures  used  for  fruit  drying. 
The  dried  product  makes  an  excellent  protein  concentrate  for  stock  or 
may  be  used  satisfactorily  in  flavoring  many  dishes  prepared  in  the 
household  and  is  suitable  for  the  preparation  of  soups.  An  extract  may 
be  made  from  yeast  and  concentrated  to  a  syrupy  consistency  as  is  meat 
extract.  The  product  has  a  flavor  similar  to  that  of  meat  extract  and 
may  be  used  for  the  same  purposes. 

Large  quantities  of  yeast  have  been  grown  by  factories  in  Germany 
primarily  for  stock  food,  using  cheap  molasses  as  a  source  of  carbo- 
hydrate for  yeast  growth  and  ammonium  sulphate  as  a  cheap  source  of 
nitrogen  for  protein  formation.  The  process  offers  a  very  quick  method 
of  producing  protein  from  inorganic  nitrogen.  A  combination  of  a 
bottom  yeast  of  the  Strept.  cerevisia  or  ellipsoideus  type  and  a  rapidly 
growing  film  yeast  of  the  Mycoderma  cerevisia  or  M.  mni  has  given  the 
greatest  yields  of  yeast  per  pound  of  raw  material  used.  Dilute  solu- 
tions yield  relatively  larger  amounts  of  yeast  than  do  more  concen- 
trated solutions. 

Sugary  solutions  from  the  hydrolysis  of  wood  may  be  used  as  a 
source  of  carbohydrate  instead  of  molasses.  In  fact,  a  variety  of  waste 
products  might  be  utilized  in  this  manner.  This  method  of  producing 
cheap  protein  possesses  great  possibilities  and  will  become  an  im- 
portant industry  it  is  believed,  as  the  world  demand  for  food  becomes 
more  acute  through  increase  of  population. 


564  MICROBIOLOGY   OF   FOODS 

BREAD* 

Success  in  bread-making  depends  more  upon  the  control  of  the  various 
types  of  fermentation  organisms  used  in  the  rising  of  the  dough  than 
upon  any  other  step  in  the  process.  Generally,  the  rising  of  the  dough, 
to  which  bread  owes  its  lightness,  is  caused  by  yeast  fermentation. 
The  yeast  is  usually  a  strain  of  Saccharomyces  cerevisice.  It  is  normally 
accompanied  by  other  organisms,  notably,  those  of  the  lactic  group. 
The  character  of  the  bread  depends  very  largely  upon  these  bacteria, 
its  quality  being  improved  by  moderate  growth  and  injured  by  ex- 
cessive growth  of  these  accessory  organisms. 

The  yeast  used  in  bread-making  is  produced  by  different  methods, 
according  to  the  custom  of  the  various  countries  or  regions.  In  the 
United  States,  compressed  yeast  prepared  as  described  elsewhere  in 
this  chapter  is  used. 

The  compressed  yeast  is  ordinarily  employed  in  one  of  two  general 
ways:  By  the  "straight  off"  method  and  as  "sponge"  or  "batter." 
In  the  former  method,  which  is  the  one  most  commonly  employed  in 
bakeries,  the  yeast,  flour,  water,  salt,  sugar,  or  sugar  substitute,  and 
shortening  (fat  or  oil)  are  mixed  at  once  and  permitted  to  rise.  Milk 
or  dried  milk  powder  is  often  used  to  replace  water  wholly  or  in  part, 
resulting  in  a  loaf  of  higher  nutritive  value  and  richer  flavor.  In  some 
cases,  proprietary  yeast  foods  are  added  with  the  other  ingredients. 

Salt  is  added  to  the  dough  to  improve  the  flavor  and  to  retard 
diastatic  and  bacterial  activities;  if  unchecked,  the  diastase  of  the 
flour  tends  to  soften  the  starch  which  gives  a  soggy  loaf  or  too  sweet  a 
flavor. 

Sugar  is  added  to  provide  material  for  yeast  fermentation — little  of 
the  sugar  is  used  for  multiplication  of  the  cells  for  the  reason  that  the 
time  of  rising  of  the  dough  is  not  sufficient  to  permit  very  great  increase 
in  number  of  the  cells  and  the  other  yeast  foods  are  not  favorable  to 
rapid  growth.  Sugar  has  been  replaced  in  practically  all  modern  baker- 
ies by  malt  syrup.  Malt  syrup  is  made  by  concentrating  to  about  80° 
Brix  in  large  copper  vacuum  pans  a  sweet  wort  made  by  the  malting 
and  mashing  of  barley  or  barley  plus  corn  or  rice  grits.  It  is  dark 
amber  in  color,  possesses  a  strong  malt  flavor,  and  usually  one  to  three 
per  cent  of  lactic  acid  made  by  "souring  of  the  mash"  with  pure  cul- 
tures of  lactic  bacteria  before  concentration.  Malt  syrup  possesses 

*  Prepared  by  W.  V.  Cruess. 


MICROBIOLOGY    OF    FERMENTED 'FOODS  565 

several  distinct  advantages  over  sugar.  The  lactic  acid  of  the  malt 
insures  a  "clean"  fermentation  in  the  dough,  improves  the  flavor  of 
the  bread,  causes  the  color  of  the  crumb  to  become  lighter,  and 
improves  the  flavor.  Unreported  data  obtained  by  the  writer  and 
experiments  reported  by  A.  Wahl*  have  shown  the  above  statements  to 
be  warranted.  Bread  made  with  malt  extract,  instead  of  sugar,  remains 
moist  for  a  longer  time  probably  because  of  the'large  amount  of  dextrin 
in  the  syrup.  The  malt  sugar  of  the  syrup  ferments  more  readily 
than  cane  sugar  and  the  syrup  is  rich  in  yeast  food,  two  factors  tending 
to  reduce  the  amount  of  yeast  necessary  and  to  improve  the  character 
of  the  fermentation.  It  is  doubtful  whether  there  is  sufficient  diastase 
in  malt  syrup  to  convert  so  great  an  amount  of  starch  into  maltose  as 
to  affect  the  yeast  fermentation,  quality,  and  texture  of  the  bread. 

It  is  probable  that  the  shortening  added  to  the  dough  does  not  affect 
the  character  of  the  fermentation;  its  principal  functions  are  to  improve 
the  texture  and  the  flavor  of  the  loaf. 

It  has  been  found  that  the  quality  of  the  bread  is  improved  and  less 
yeast  is  required  if  a  small  amount  of  plaster  of  Paris,  calcium  sulphate, 
is  added  to  the  flour.  Ammonium  sulphate  possesses  a  remarkable 
stimulating  effect  upon  the  yeast.  If  small  quantities  of  this  salt  are 
used  it  all  disappears  during  the  rising  of  the  dough.  It  is  assumed  that 
it  is  taken  up  by  the  yeast  and  converted  into  protein.  One  proprietary 
mixture  used  by  bakers  consists  principally  of  a  mixture  of  ammonium 
sulphate  and  plaster  of  Paris. 

The  amount  of  water  added  will  vary  with  the  strength  of  the  flour, 
weak  flours  requiring  less  and  giving,  therefore,  a  smaller  weight  of 
bread  than  strong  flours. 

The  flour  and  other  ingredients  are  brought  to  a  definite  favorable 
temperature,  25°  to  30°,  before  mixing  with  the  yeast.  The  dough 
mixing  room  in  modern  plants  is  maintained  at  a  constant  temperature 
and  high  humidity  by  means  of  a  thermostat  and  hygrometer  controlled 
heating  and  ventilating  system.  The  latter  consists  of  a  large  fan, 
heating  coils,  air  humidifying  chamber,  and  air-distributing  pipes.  A 
high  humidity  is  necessary  to  prevent  the  surface  of  the  dough  drying 
out  during  its  stay  in  the  mixing  room.  A  constant  temperature 
is  necessary  to  insure  that  the  bread  will  rise  on  "schedule"  time  if 
other  conditions  are  carefully  controlled.  It  is  usual  to  mix  the  dough, 

*  Wahl,  A.,  Journal  of  Industrial  and  Engineering  Chemistry,  1915,  page  773. 


566  'MICROBIOLOGY  OF.  FOODS 

knead  the  rising  dough, ' 'proof"  the  loaves,  and  bake  the  bread  within 
a  regular  schedule  of  eight  hours  or  less. 

Mixing  of  the  ingredients  must  be  thorough  to  insure  uniform 
fermentation  throughout  the  dough.  Mixing  also  results  in  the  inclu- 
sion of  air  with  the  dough  which  stimulates  yeast  growth  and  activity. 
During  the  rising  of  the  dough  several  changes  take  place.  Carbon 
dioxide  is  formed  and  distends  the  dough  by  the  formation  of  small 
bubbles  of  gas  held  by  the  gluten  of  the  flour.  Wheat  flour  is  the  only 
one  possessing  enough  gluten  to  permit  of  this  phenomenon.  The 
proteins  of  the  flour  are  to  some  extent  peptonized  by  proteolytic 
enzymes  of  the  flour,  thus  tending  to  reduce  the  harsh  texture  of  the 
raw  dough.  Diastase  is  active  to  a  slight  extent  and  also  tends  to 
soften  the  texture  of  the  dough.  Lactic  and  other  bacteria  develop 
to  an  extent  depending  largely  upon  the  time  and  upon  the  original 
number  of  such  organisms  present.  Contrary  to  popular  belief,  lactic 
organisms  are  not  the  ones  usually  responsible  for  the  "sour  taste" 
of  some  breads,  this  undesirable  flavor  is  usually  due  to  butyric  acid 
organisms. 

Ordinarily  the  dough  is  allowed  to  rise  in  shallow  wooden  troughs 
in  the  dough  mixing  room.  After  it  has  risen  sufficiently  it  is  "cut 
down"  and  rekneaded.  Often  the  cutting  and  rekneading  are  again 
repeated  before  the  bread  is  molded.  The  cutting  down  prevents  the 
bread  rising  too  rapidly  and  permits  a  sufficiently  long  period  of  rising 
to  insure  the  proper  flavor  and  texture,  and  the  frequent  kneading 
results  hi  the  formation  of  a  loaf  of  more  uniform  texture.  Too  much 
yeast  causes  the  dough  to  develop  large  gas  pockets  or  gas  may  escape 
from  the  dough  from  large  bubbles,  resulting  in  the  breaking  and  slacken- 
ing of  the  dough.  Too  little  yeast  results  in  too  slow  a  fermentation 
with  consequent  increase  in  danger  from  growth  of  undesirable  organisms. 
The  dough  from  the  dough  or  "proof"  room  goes  to  mechanical 
molding  machines  which  form  the  dough  into  loaves  of  the  desired 
weight  and  deliver  them  to  the  proof  box  where  the  loaves  are  subjected 
to  a  temperature  favorable  to  rapid  fermentation.  They  may  or  may 
not  be  rekneaded  before  baking. 

Baking  is  in  many  large  plants  accomplished  in  "traveling  ovens" 
in  which  the  loaves  traverse  the  oven  on  heavy  metallic  conveyors, 
thus  resulting  in  a  great  saving  of  labor  and  in  standardization  of 
baking  time.  The  ovens  are  electrically  controlled  by  thermo-regula- 


MICROBIOLOGY   OF   FERMENTED    FOODS  567 

tors.  Other  bakeries  use  stationary  ovens  of  various  types  and  handle 
the  bake  pans  by  hand  labor. 

During  baking,  the  gas  pockets  in  the  dough  expand  with  the 
heat  and  increase  the  size  of  the  loaf.  The  gluten  is  coagulated  by 
the  heat  and  thus  retains  the  size  and  shape  of  the  gas  bubbles  at  the 
moment  of  coagulation.  The  yeast  activity  is  for  a  short  time  stimu- 
lated but  the  cells  are  killed  when  the  dough  reaches  60°.  Peptic 
activity  is  for  a  time  stimulated,  resulting  in  some  softening  of  the 
gluten.  Diastase  increases  in  activity  until  65°  to  70°  is  reached  and 
then  decreases  until  the  temperature  is  reached  which  destroys  this 
enzyme.  It  hydrolyzes  or  gelatinizes  some  of  the  starch  on  the  surface 
of  the  loaf.  Heat  dextrinizes  part  of  the  starch  in  this  locality  also,  and 
the  drying  out  of  this  layer  of  dextrinized  starch  gives  the  crust.  The 
interior  of  the  loaf  probably  does  not  reach  a  temperature  much  above 
1 00°  during  baking  because  the  evaporation  of  water  maintains  the 
dough  at  the  boiling  point  of  water.  The  carbon  dioxide  and  alcohol 
formed  during  fermentation  are  driven  off  although  some  of  the  less 
volatile  esters  and  organic  acids  formed  by  yeast  or  bacteria  remain  in 
the  bread  to  give  it  its  characteristic  flavor. 

Bread  made  by  this  method  should  have  a  flinty  nut-brown 
crust  which  cracks  when  broken;  the  crumb  should  be  porous,  not  soggy, 
free  from  large  gas  pockets,  of  even  fine  grained  texture  and  elastic; 
the  flavor  should  be  clean  and  sweet  and  the  color  white  or  creamy 
white.  It  should  not  possess  a  disagreeable  yeasty  or  butyric  odor. 
A  yeasty  odor  usually  comes  from  stale  yeast — fresh  clean  yeast  can 
be  used  in  very  large  quantities  without  imparting  a  yeasty  odor  or 
flavor. 

In  the  ''sponge  dough"  method  commonly  used  by  housewives  and 
by  some  bakers  a  thin  batter  of  yeast,  flour,  and  other  ingredients 
is  prepared  on  the  day  before  the  dough  is  to  be  made.  Potato  flour  or 
grated  potato  is  often  added  to  furnish  yeast  food.  Malt  syrup  is  used 
for  the  same  purpose.  Vigorous  fermentation  and  probably  consider- 
able growth  of  yeast  occurs  in  this  batter  which  is  softened 
through  peptic  and  diastatic  activity  and  considerable  opportunity  for 
bacterial  growth  is  given.  After  about  twenty-four  hours  standing  it  is 
mixed  with  flour  and  handled  as  described  above  in  the  straight  dough 
method  except  that  usually  only  one  "cutting  down"  of  the  dough  is 
employed.  Bread  made  in  this  way  usually  possesses  a  more  agreeable 


568  MICROBIOLOGY   OF  "FOODS 

flavor  and  texture  than  bread  made  by  the  straight  method  and  the 
bread  remains  fresh  for  a  longer  period.  Where  dry  yeast  cakes  are 
used,  the  sponge  method  is  to  be  preferred. 

In  many  European  countries  a  "sour  dough"  starter  is  used 
to  leaven  the  loaf.  According  to  the  French  method  described 
by  Boutroux*  a  little  of  the  dough  ready  for  baking  is  set  aside  and 
mixed  with  flour  and  water  and  permitted  to  stand  four  or  five  hours. 
This  operation  is  repeated  several  times  before  the  "leaven"  is  ready 
for  mixing  with  the  dough.  The  repeated  addition  of  flour  invigorates 
the  yeast  by  supplying  it  with  food,  thus  maintaining  active  yeast 
growth,  which  tends  to  discourage  the  growth  of  less  desirable  ferments. 
Fresh  dough  with  about  one-third  its  bulk  of  leaven  is  mixed  for  the  final 
baking  and  fermentation  is  allowed  to  proceed  again  -for  a  short  time. 
In  spite  of  the  repeated  additions  of  flour  during  preparation  of  the 
leaven  there  is  a  rapid  growth  of  lactic  and^other  bacteria  resulting  in 
the  development  of  the  acid  and  characteristic  flavor  of  genuine 
French  bread.  The  long  fermentation  results  in  considerable  softening 
of  the  gluten,  causing  large  holes  to  form  in  the  dough,  and  in  marked 
diastasic  action  which  also  affects  the  texture  of  the  loaf.  Imitation 
"French  bread"  as  usually  sold  in  the  United  States  is  made  with 
compressed  yeast  and  does  not  resemble  true  French  bread  in  flavor. 

Sour  dough  breads  are  common  in  Italy  and  southern  Europe. 
Usually  the  fermentation  is  less  carefully  controlled  than  in  France, 
imparting  a  more  acid  and  to  the  American  taste,  a  less  agreeable  flavor. 

In  some  localities  an  impure  natural  yeast  is  prepared  for  bread 
making.  Flour,  hops,  hot  water,  and  ground  malt,  are  mixed  together, 
and  allowed  to  stand  until  the  diastase  converts  most  of  the  starch  to 
sugar.  The  sweet  liquid  is  drawn  off  and  allowed  to  ferment.  The 
yeast  so  grown  is  known  as  "  virgin  barm."  If  a  starter  of  yeast  from  a 
previous  lot  is  added,  the  yeast  is  then  termed  "Parisian  barm."  The 
sweet  liquid,  according  to  Jago,  is  of  about  14°  Brix  when  freshly 
prepared.  The  hops  serve  to  check  the  growth  of  undesirable  bacteria. 
Nevertheless,  the  barm  is  a  mixture  of  several  yeast  varieties  and 
bacteria. 

The  fermented  liquid  and  yeast  are  mixed  with  the  flour  in  making 
up  the  dough. 

The  Mexican  tortilla  contains  no  leaven  and  is  baked  as  soon  as 

*  Boutroux,  L.     Le  pain  et  la  panification. 


MICROBIOLOGY    OF   FERMENTED    FOODS  569 

the  flour,  water  and  salt  are  mixed  and  thoroughly  beaten.  Similar 
breads  are  made  in  other  primitive  countries. 

Salt-rising  bread  has  been  studied  from  the  bacteriological  stand- 
point by  Kohman.  *  A  starter  is  prepared  by  scalding  corn  meal  with 
hot  milk.  Salt  is  added  to  the  mixture.  The  mixture  is  kept  in  a  warm 
place  until  in  active  fermentation  and  is  then  mixed  with  wheat  flour 
and  salt  to  give  a  dough  of  normal  consistency.  The  organisms  are  of  a 
spore-bearing  type  which  readily  survives  a  temperature  of  75°,  while 
B.  coli  and  other  undesirable  organisms  are  eliminated.  The  organisms 
are  furnished  by  the  corn  meal.  These  were  purified  by  Kohman  who 
found  that  they  lost  their  gas-producing  power  when  preserved  in  or  on 
usual  laboratory  media,  but  retained  their  desirable  properties  includ- 
ing gas  production  if  preserved  in  the  dry  state  after  mixing  with  a 
starchy  material.  The  gases  formed  during  salt  rising  bread  fermenta- 
tion were  about  two-thirds  hydrogen  and  one-third  carbon  dioxide. 
Yeast  is  not  of  importance  in  the  rising  of  this  bread. 

Bread  is  subject  to  a  number  of  imperfections  or  diseases.  Soggy 
bread  may  be  due  to  a  prolonged  period  of  rising,  use  of  insufficient 
yeast  or  poor  yeast,  use  of  too  much  water  in  proportion  to  the  flour, 
or  too  weak  flour,  insufficient  kneading  or  improper  baking.  Ropy 
or  slimy  bread  is  caused  by  spore-bearing  organisms,  usually  B.  mesen- 
tericus  vulgatus,  which  live  through  the  baking  process.  Either  the 
yeast  or  the  flour  may  be  the  source  of  infection.  It  is  stated  that 
bread  becomes  stale  because  of  the  migration  of  moisture  from  the 
crumb  to  the  crust,  leaving  the  crumb  dry  and  crumbly  and  the  crust 
pliable  and  soft  instead  of  flinty  and  brittle.  It  is  also  due  in  part  to 
the  starch  reverting  from  the  semi-soluble  form  of  the  fresh  loaf  to  its 
original  harsh  form.  Molds  of  the  mucor  group  sometimes  develop 
quickly  in  a  warm  moist  atmosphere. 

Sour  bread,  the  most  dreaded  of  all  imperfections,  is  usually  due  to 
the  growth  of  butyric  organisms.  The  clean  sour  flavor  developed  by 
lactic  bacteria  is  not  so  objectionable,  and  is  even  preferred  to  the 
usual  flavor  of  bakers'  bread  by  many. 

Micrococcus  prodigiosus  may  develop  on  moist  bread  after  long 
standing,  with  the  formation  of  red  spots.  It  seldom  occurs,  however, 
and  is  not  considered  harmful  to  health. 

*  H.  A.  Kohman.  Salt  Rising  Bread  and  Some  Comparisons  with  Bread  made  by  Yeast. 
Jour.  Ind.  and  Eng.  Chem.,  1912,  pp.  20,  100. 


570  MICROBIOLOGY   OF   FOODS 

PRESERVATION  OF  VEGETABLES  BY  FERMENTATION* 

Lactic  acid  is  an  excellent  preservative  and  affords  a  very  common 
means  of  preserving  certain  kinds  of  vegetables.  The  most  familiar 
examples  are  sauerkraut  and  dill  pickles.  In  some  European  countries 
many  kinds  of  vegetables  are  preserved  by  lactic  or  "kraut"  fermenta- 
tion—this method  replacing  to  a  large  extent  preservation  by  can- 
ning. String  beans,  greens  of  all  sorts,  cauliflower,  carrots,  turnips,  and 
beets  are  very  satisfactorily  preserved  in  this  way;  peas  tend  to  become 
rancid  in  flavor. 

Two  general  fermentation  methods  are  used,  dry-salting  and 
brine  For  juicy  vegetables,  such  as  cabbage  and  greens,  the  dry-salt- 
ing method  is  preferable;  for  large  vegetables  or  vegetables  of  less  water 
content  fermentation  in  brine  is  advisable.  Sufficient  salt  is  sometimes 
used  to  prevent  all  fermentation. 

In  the  preparation  of  sauerkraut  the  dry-salting  process  is  used. 
The  heads  of  cabbage  are  cored,  the  outer  leaves  are  removed,  and  the 
remainder  is  cut  into  thin  shreds.  For  each  100  pounds  of  cabbage 
2^  pounds  of  salt  is  added  and  mixed  with  the  cabbage  in  open  wooden 
containers  varying  in  size  from  a  small  barrel  or  crock  for  home  use  to 
tanks  holding  a  thousand  gallons  or  more  in  large  factories.  Pressure 
is  applied  to  the  mixture,  by  weights  in  the  household  and  by  screw  or 
beam  presses  in  factories.  The  osmotic  action  of  the  salt  combined 
with  the  pressure  forces  the  juice  from  the  cabbage.  In  this  juice 
a  very  vigorous  gaseous  and  acid  fermentation  ensues  in  which  the 
mannite  and  sugars  of  the  cabbage  are  converted  into  lactic  acid, 
acetic  acid,  alcohol,  succinic  acid  (and  in  some  cases  butyric  acid). 
Carbon  dioxide,  hydrogen,  methane  and  various  aromatic  esters  and 
other  bodies  are  also  formed.  At  the  same  time  the  protein  is  de- 
composed more  or  less.  Without  the  addition  of  salt  a  putrefactive 
fermentation  is  apt  to  result;  the  salt  to  this  degree  acts  as  a  governor 
of  the  types  of  fermentation  that  occur.  It  tends  to  favor  lactic  fer- 
mentation and  check  putrefaction.  The  fermented  product  owes 
its  keeping  qualities  to  the  lactic  acid  formed  during  the  fermentation 
period.  The  amount  of  acid  formed  is  usually  from  0.5  to  1.5  per  cent. 

Mycoderma  vini  and  molds  develop  rapidly,  especially  in  warm 
weather,  on  the  surface  of  the  fermented  liquid  and  rapidly  "devour" 

*  Prepared  by  W.  V.  Cruess. 


MICROBIOLOGY   OF   FERMENTED   FOODS  571 

the  lactic  acid.  This  soon  results  in  a  reduction  of  the  acidity  to  the 
point  where  putrefactive  organisms  may  develop.  These  acid-de- 
stroying organisms  are  aerobic.  Therefore,  prevention  of  their  growth 
consists  simply  in  excluding  air  from  the  fermented  material.  Tanks 
or  barrels  may  be  filled  with  dilute  brine  and  headed  up;  small  con- 
tainers are  sealed  with  paraffin  or  vegetable  oil  to  exclude  air.  The 
sealing  of  the  container  is  not  done  until  fermentation  is  complete. 
In  warm  weather  complete  fermentation  will  take  place  in  eight  to  ten 
days;  in  cool  weather  two  to  four  weeks  is  required.  Mycoderma 
grows  very  rapidly  in  warm  weather  forming  a  heavy  gray  wrinkled 
film;  in  cold  weather  it  develops  very  slowly  or  not  at  all;  hence  more 
precautions  against  its  development  must  be  taken  during  the  summer 
months  than  during  the  fall  or  early  spring. 

Weiss  has  isolated  some  sixty-five  different  species  of  bacteria  from 
sauerkraut,  most  of  which  were  indifferent  or  harmless.  The  writer 
can  see  no  reason  why  Bacillus  botulinus  might  not  develop  in  cabbage 
or  other  vegetables  during  or  following  fermentation  and  suggests  this 
as  a  good  field  for  investigation  by  the  various  laboratories  at  present 
engaged  in  the  investigation  of  B.  botulinus  in  its  relation  to  food 
preservation. 

String  beans  and  many  varieties  of  greens  may  be  preserved  in  the 
same  manner  as  sauer  kraut.  The  best  results  are  obtained  if  a  small 
amount  of  vinegar  is  added  to  the  brine  to  favor  lactic  organisms  and  to 
discourage  putrefactive  forms. 

In  the  manufacture  of  most  pickles,  the  raw  material  (cucumbers, 
cauliflower,  etc.)  undergoes  a  lactic  acid  fermentation  before  receiving 
the  final  vinegar  or  other  preservative  solution  or  sauce.  Normally, 
the  cucumbers  are  first  placed  in  a  brine  of  about  45°  salometer  test 
(about  1 2  per  cent  sodium  chloride)  in  open  vats.  As  osmotic  action 
and  fermentation  proceed,  the  brine  becomes  diluted  by  the  vegetable 
juices;  if  the  concentration  falls  much  below  45°  salometer,  putrefac- 
tion and  softening  occur.  An  attempt  is  therefore  made  to  increase 
the  concentration  to  60°  salometer  test  (15  to  17  per  cent  sodium  chlor- 
ide) by  progressive  additions  of  salt.  The  vegetables  are  well  covered 
by  the  brine.  Vigorous  lactic  acid  fermentation  occurs  in  the  brine 
and  vegetables.  Mycoderma  mni  and  film-forming  bacteria  develop 
at  the  surface  of  the  liquid  but  are  usually  not  sufficiently  active  in  the 
60°  brine  to  reduce  appreciably  the  acidity.  Cucumbers  are  often 


572  MICROBIOLOGY   OF.  FOODS 

held  a  year  or  more  in  this  way.  Subsequent  pickling  operations  con- 
sist in  leaching  out  the  excess  salt  in  warm  or  hot  water,  firming  the 
flesh  with  a  dilute  solution  of  alum  and  impregnation  of  the  cucumbers 
with  plain  or  spiced  or  sweetened  vinegar. 

Dill  pickles  are  prepared  by  fermentation  of  cucumbers  in  a 
40°  to  45°  brine  (10  to  12  per  cent  sodium  chloride)  in  closed  barrels 
fitted  with  small  vent  holes  for  escape  of  gas.  Dill  and  other 
herbs  and  spices  are  packed  in  the  brine  with  the  cucumbers  before 
fermentation.  Enough  acid  is  formed  to  preserve  the  pickles  indefi- 
nitely if  air  is  excluded.  Often  the  finished  pickles  are  pasteurized  in 
lacquered  cans  for  domestic  use.  The  high  acidity  renders  the  steriliza- 
tion very  easily  accomplished. 

Cauliflower,  peppers,  ears  of  sweet  corn,  and  other  vegetables  have 
been  held  successfully  in  brines  of  10  to  12  per  cent  salt  (40°  to  48° 
salometer  test),  although  the  products  possess  a  distinct "  kraut "  flavor. 
If  vegetables  of  any  variety  are  mixed  with  25  to  33  per  cent  of 
their  weight  of  salt  Jthey  may  be  preserved  indefinitely  without  fer- 
mentation, provided  they  are  sealed  in  barrels  or  jars  or  under  parafime 
to  prevent  evaporation  of  moisture.  Corn  and  string  beans  are  excel- 
lent so  preserved.  The  process  offers  a  home  method  of  preserving 
vegetables  without  danger  from  botulism.  Where  dry  salt  can  not  be 
employed  the  vegetables  may  be  packed  in  a  saturated  solution  of  salt. 
The  salt  preserved  products  must  be  soaked  in  water  before  use  to  re- 
move excess  of  sodium  chloride. 

THE  RELATION  OF  BACTERIA  TO  OLIVE  PICKLING  AND  CANNING* 

The  green  olive  of  commerce  is  a  fermented  product  preserved  by 
the  lactic  acid  formed  during  the  pickling  process.  The  process  used 
dates  from  antiquity  and  is  the  result  of  long  years  of  experience  and 
slow  development. 

Olives  of  full  size  but  still  immature  are  used.  The  Queen  olive 
of  commerce  is  principally  of  the  Sevillano  variety.  The  small 
green  olives  are  usually  of  the  Manzanillo  variety,  although  other 
types  are  often  used. 

The  fruit  is  first  placed  in  a  lye  containing  about  2  per  cent  of 
sodium  hydroxide  which  is  allowed  to  penetrate  the  fruit  almost 
to  the  pit.  The  lye  is  then  removed  and  replaced  with  cold  water 

*  Prepared  by  W.  V.  Cruess. 


MICROBIOLOGY    OF   FERMENTED   FOODS  573 

which  is  frequently  changed  until  the  fruit  is  practically  free  from  lye. 
The  lye  hydrolyzes  the  amygdalin,  a  bitter  principle  of  the  olive. 

The  olives  are  then  placed  in  barrels  and  the  barrels  filled  with  a 
brine  of  7  to  10  per  cent.  (28°  to  40°  salometer)  salt  solution  and  rolled 
into  a  warm  room  or  the  warm  sunshine  to  undergo  spontaneous  fer- 
mentation. Many  types  of  organisms  develop  but  the  high  salt  con- 
centration favors  the  growth  of  lactic  bacteria.  Six  weeks  to  two 
months  is  necessary  for  the  fermentation.  Air  must  be  excluded 
to  prevent  the  growth  of  acid-destroying  organisms  and  to  prevent 
browning  of  the  color  of  the  olives  by  oxidation.  The  finished  olives 
are  packed  in  glass  containers  without  sterilization.  The  acidity 
of  the  fruit  is  relied  upon  to  preserve  the  product.  The  characteristic 
flavor  of  green  olives  is  due  to  fermentation.  Often,  however,  various 
decompositions  occur  in  the  jar  resulting  in  softening  of  the  fruit  and  the 
development  of  a  disagreeable  flavor  and  odor.  B.  coli  is  a  common 
offender  in  this  regard.  The  writer  believes  that  a  much  more  health- 
ful and  sanitary  product  would  be  obtained  if  the  bottled  olives  were 
pasteurized. 

Ripe  olives  are  packed  extensively  in  California  in  cans.  The 
finished  product  is  dark  brown  or  black  in  color  and  neutral  or 
slightly  alkaline  in  reaction.  The  usual  process  consists,  first,  in  plac- 
ing the  ripe  fruit  in  barrels  or  tanks  filled  with  5  to  7  per  cent,  brine. 
In  these  containers,  a  vigorous  gaseous  and  lactic  acid  fermentation 
takes  place  for  about  two  weeks  and  a  film  of  yeast-like  cells  accumu- 
lates at  the  surface  in  many  cases.  Some  factories  omit  this  prelimin- 
ary fermentation  but  those  who  employ  it  claim  that  it  renders  the  olives 
firmer,  porous,  easily  penetrated  by  the  lye  used  in  pickling,  and  of 
superior  flavor.  The  writer's  experience  leads  him  to  believe  that  the 
fermentation  process  has  little  merit  and  may  be  one  source  of  infection 
of  olives  with  B.  bolulinus. 

The  olives  are  treated,  in  the  usual  process,  with  dilute  lye  % 
to  2  per  cent  sodium  hydroxide  for  a  period  of  time  sufficient 
to  permit  the  lye  to  penetrate  the  skin  of  the  fruit  and  a  short  distance 
into  the  flesh.  They  are  then  exposed  to  the  air  or  are  submitted  to  a 
stream  of  compressed  air  in  water  to  darken  the  color.  The  polyphenols 
of  the  olive  flesh  rapidly  oxidize  to  a  black  color  in  the  presence  of  air 
and  dilute  sodium  hydroxide.  The  darkened  fruit  is  given  a  second 
lye  weaker  than  the  first  until  the  lye  reaches  the  pit.  This  destroys 


574  MICROBIOLOGY   OF   FOODS 

the  bitter  principle.  The  lye  is  removed  by  repeated  leaching  of  the 
fruit  with  water.  In  former  practice  it  was  then  covered  with  a  dilute 
brine  of  about  2  per  cent  sodium  chloride  which  was  gradually  increased 
to  5  per  cent.  During  this  "salt  curing"  process  of  two  to  six  weeks 
opportunity  was  afforded  for  a  great  variety  of  bacteria  to  develop. 
B.  coli  could  be  found  very  frequently.  Often  gaseous  fermentation 
of  the  fruit  developed  or  the  surface  of  the  olives  became  slimy  through 
the  growth  of  mold,  bacteria,  and  even  amoebae  or  the  fruit  softened 
through  putrefactive  organisms.  Most  factories  now  eliminate  or 
greatly  shorten  the  brining  process  to  avoid  the  bacterial  changes  noted 
above. 

The  olives  are  canned  in  a  3  to  5  per  cent  (12°  to  20°  salometer) 
salt  solution  and  sterilized  at  212°  to  25o°F.,  depending  upon  the 
factory.  Recent  investigations  by  the  writer  have  shown  that  olives 
heated  to  2i2°F.  only  in  cans  are  not  sterile  but  in  practically  all  cases 
contain  living  spore-bearing  organisms.  Temperatures  of  230°  to 
24o°F.  may  be  used  for  thirty  to  forty  minutes  without  seriously  in- 
juring the  quality  of  the  fruit. 

Recently  several  cases  of  botulism  from  commercially  canned 
olives  and  one  from  green  olives  have  been  reported.  Three  factories 
were  represented  in  these  outbreaks.  In  all  cases  the  olives  have  been 
processed  in  the  cans  at  2 1 2°F.  only.  No  cases  have  occurred  from  this 
fruit  sterilized  under  steam  pressure.  Cases  have  been  most  frequent 
from  fruit  canned  in  glass  because  of  the  difficulty  of  sterilizing  glass 
containers  under  pressure  without  breakage.  Dr.  E.  C.  Dickson  of 
Stanford  University,  Dr.  K.  F.  Meyer  of  the  University  of  California, 
and  Dr.  M.  J.  Rosenau  are  engaged  upon  an  extensive  and  detailed 
study  of  the  death  temperatures  and  other  properties  of  B.  botulinus 
strains  from  food  poisoning  cases.  Their  work  is  under  a  grant  from 
the  National  Canners  Association,  given  as  a  direct  result  of  the  out- 
breaks from  canned  olives. 

As  now  canned,  ripe  olives  are  safe  because  of  effective  sterilization 
methods. 

SILAGE  FERMENTATION* 

The  character  of  the  changes  brought  about  in  silage  varies  with  the 
material  used  in  filling  the  silo  and  with  the  method  of  filling.  Beet 
cosettes  in  silos  undergo  alcoholic  and  lactic  fermentation,  very  large 

*  Prepared  by  W.  V.  Cruess. 


MICROBIOLOGY    OF    FERMENTED    FOODS  575 

losses  of  sugar  occurring  through  fermentation  processes.  Often  3 
per  cent  or  more  of  lactic  acid  is  formed,  beet  pulp  silage  in  this  respect 
representing  one  of  the  most  acid  types  of  silage  commonly  produced. 
The  proteins  of  the  beets  also  undergo  decomposition  to  a  limited  ex- 
tent with  consequent  reduction  of  feeding  value.  Beet  cosettes  are 
now  dried  in  most  modern  mills  because  of  the  large  losses  of  feeding 
value  in  beet  silage  and  because  of  the  better  keeping  quality  of  the 
dried  product. 

Pea  vine  and  other  highly  nitrogenous  materials  tend  to  undergo 
putrefaction  rather  than  lactic  fermentation  but  make  excellent  silage 
when  combined  with  corn  or  other  starchy  materials. 

The  fermentation  of  corn  silage  was  first  systematically  studied 
by  Burrill*  who  found  that  the  hot  fermentations  sometimes  encount- 
ered in  silage  were  frequently  induced  by  slow  filling  of  the  silo,  thus 
permitting  oxidizing  thermophilic  organisms  to  develop.  Silage  near 
the  surface  of  the  filled  silo  often  reaches  high  temperatures  for  this 
same  reason.  Temperatures  of  60°  to  70°  are  not  uncommon. 

In  the  normal  fermentations  of  silage,  however,  the  temperature 
in  the  depths  of  the  silo  seldom  exceeds  35°,  permitting  the  growth  of  a 
varied  microflora  in  which  lactic  organisms  predominate.  Acetic  acid 
and  propionic  acids  have  been  shown  by  Dox  and  Neidig  of  Iowa  to 
be  regular  constituents  of  corn  silage.  Their  results  indicate  that  a 
small  amount  of  butyric  acid  is  to  be  found  in  normal  silage  and  that 
this  often  increases  in  silage  of  poor  quality.  Formic  acid  was  found 
in  a  few  samples.  Ethyl  alcohol  and  propyl  alcohol  occur  in  deter- 
minable  quantities  and  higher  alcohols  in  traces.  It  is  believed  that  the 
alcohols  are  formed  as  a  result  of  bacterial  rather  than  yeast  fermenta- 
tion as  the  yeasts  found  in  silage  are  usually  of  the  Mycoderma  group 
rather  than  of  the  alcohol-forming  types. 

Silage  resembles  sauerkraut  in  many  respects,  although  the  fact  that 
silage  is  less  watery  than  sauerkraut  modifies  the  character  of  the  or- 
ganisms and  the  intensity  of  the  fermentation.  Corn  silage  normally 
contains  more  than  i  per  cent  total  acid. 

So-called  "sweet  silage"  may  be  caused  in  some*  cases  by  thermo- 
philic fermentation  which  reaches  a  high  enough  temperature  to  destroy 
the  lactic  bacteria  or  in  other  cases  by  the  use  of  material  too  low  in 
sugar  to  afford  enough  of  this  compound  for  appreciable  lactic  acid 

*Burrill,  T.  J.,  Biology  of  Silage.     Bulletin  7.     Illinois  Station,  1889. 


57^  MICROBIOLOGY   OF   FOODS 

formation.  "Sweet  silage"  always  contains  an  appreciable  amount 
of  acid. 

According  to  Hunter  and  Bushnell  of  the  Kansas  Station*  four 
groups  of  organisms  are  responsible  for  the  changes  occurring  in  the 
silo:  (i)  the  acid  group,  (2)  the  colon  group,  (3)  yeasts,  and  (4)  miscel- 
laneous. They  found  that  the  most  important  acid  formers  belong 
to  the  bulgarian  group.  Recently  the  use  of  cultures  of  B.  bulgaricus 
grown  in  milk  as  starters  for  silage  fermentations  has  been  advocated 
in  farm  journals.  Whether  this  practice  will  prove  successful  and 
desirable  remains  to  be  seen,  but  it  would  certainly  appear  to  be  a  logical 
procedure  for  the  reason  that  the  addition  of  lactic  cultures  to  beet 
cosette  silage  has  proved  successful. 

Probably  a  great  deal  of  the  gas  formation  in  silage  is  due  to  mem- 
bers of  the  colon  group. 

Plant  enzymes  are  of  some  importance  in  silage  fermentation. 
Some  of  the  starch  of  corn  or  corn  stalks  is  converted  into  "sugar  by  the 
plant  diastase  and  the  presence  of  a  plant  invertase  which  acts  upon  the 
cane  sugar  present  has  been  demonstrated.  However,  it  is  believed  that 
no  appreciable  proportion  of  the  rise  in  temperature  in  the  silo  is  due  to 
these  or  other  similar  purely  plant  activities.  It  is  held  by  most 
investigators  that  the  important  changes  are  due  rather  to  bacterial 
activity. 

Recently  the  work  of  Graham  of  Kentucky  and  others  has  brought 
to  light  in  a  startling  manner  the  importance  of  Bacillus  botulinus  in 
relation  to  forage  poisoning  from  silage  and  hay  or  straw.  Because  of 
the  fact  that  hay  or  silage  responsible  for  stock  poisoning  was  usually 
moldy  it  was  assumed  that  the  mold  was  the  responsible  agent.  Silage 
at  the  surface  of  the  silo  becomes  moldy  and  alkaline  in  reaction  and  is 
considered  by  many  stock  men  as  poisonous  because  of  the  mold. 

It  has  now  been  demonstrated,  however,  that  the  most  common 
death-producing  organism  in  silage  is  very  similar  if  not  identical  with 
B.  botulinus,  Pearson  as  early  as  1900  demonstrated  the  relation 
between  a  sporadic  outbreak  of  forage  poisoning  and  the  poisonous 
quality  of  ensilage  fed  to  the  stock  in  question.  Stonge  and  Buchanan 
of  Iowa  obtained  positive  results  under  conditions  similar  to  the  above. 
More  recently  Bush  and  Gridley  of  Illinois  and  Graham,  Brueckner 
and  Pontius  of  Kentucky,  have  shown  B.  botulinus  or  an  organism 

*  Kansas  Station  Technical  Bulletin  2  (1916). 


MICROBIOLOGY   OF   FERMENTED   FOODS  577 

closely  resembling  this  organism  to  be  responsible.  Hart  of  the 
University  of  California  has,  late  in  1919,  studied  several  outbreaks  of 
this  character  in  California.  He  finds  that  the  number  of  forage  poison- 
ing cases  increases  during  the  winter,  probably  because  sufficient  tune 
has  elapsed  since  the  preceding  harvest  to  permit  formation  of  toxin 
in  silage  or  hay. 

To  date,  no  definite  method  of  preventing  the  growth  of  this  organ- 
ism in  silage  has  been  developed,  although  its  growth  is  undoubtedly 
influenced  by  the  composition  of  the  silage  and  the  character  of  the 
fermentations  taking  place  in  the  product.  It  would  appear  logical 
to  induce  vigorous  lactic  fermentation  in  the  silage  in  order  to  check 
the  development  of  B.  botulinus. 

Hart  of  California  found  the  two  groups,  namely,  A  and  B  strains, 
of  the  organism  in  forage  poisoning  cases  and  confirmed  the  work  of 
others  that  the  antitoxin  from  an  A  strain  does  not  protect  against 
the  toxin  of  a  B  strain  and  vice  versa.  He  has  not  had  any  notable 
success  in  preventing  the  death  of  animals  already  showing  symptoms 
of  botulism  by  the  use  of  antitoxin  but  has  obtained  protection  where 
the  antitoxin  is  administered  with  the  toxin  or  very  shortly  afterward. 
He  has  succeeded  in  immunizing  horses  to  very  heavy  doses  of  the 
toxin  and  has  thereby  obtained  the  antitoxin  in  quantity. 

The  problems  presented  are  extremely  serious  and  important  and 
well  worthy  the  attention  of  all  investigators  in  states  which  use  the 
silo  extensively.  B.  botulinus  has  of  late  intruded  itself  upon  our  atten- 
tion in  many  ways  and  its  control  offers  one  of  the  most  important 
problems  confronting  the  bacteriologists  of  this  country. 

MALT  SYRUPS* 

Several  types  of  malt  syrup  are  produced  commercially  in  the 
United  States.  The  most  common  form  is  that  used  by  bakeries  in 
bread  making.  In  preparing  this  syrup  kiln  dried  malt  is  ground, 
mixed  with  water  and  mashed,  first  at  a  temperature  of  50°  to  52°  to 
favor  lactic  fermentation,  and  later  at  60°  or  higher  to  convert  the  re- 
maining starch  into  maltose  and  dextrin.  The  resulting  liquid  is 
strained  or  filtered  and  is  concentrated  in  vacuo  to  about  78°  Balling. 
One  of  the  most  important  constituents  of  the  syrup  is  the  i  to  3  per 
cent,  of  lactic  acid  content.  This  is  usually  formed  by  selected  strains 

*  Prepared  by  W.  V.  Cruesg. 
37 


57$  MICROBIOLOGY    OF    FOODS 

of  lactic  organisms  during  the  mashing  process.  In  some  factories, 
the  lactic  fermentation  is  carried  out  in  a  special  tank  separate  from 
the  general  mash  tank;  this  acidified  liquid  is  then  added  to  the  sacchar- 
ified liquid  from  the  mash  tank.  This  method  greatly  simplifies  the 
mashing  of  the  malt  used  for  the  main  bulk  of  the  syrup,  making  pre- 
liminary mashing  at  50°  to  52°  unnecessary. 

"Malt  glucose"  or  refined  malt  syrup  is  a  colorless  syrup  resembling 
ordinary  refined  corn  syrup  in  appearance  but  is  sweeter  and  of  richer 
flavor.  It  is  usually  prepared  by  saccharifying  gelatinized  corn  or 
rice  starch  with  barley  malt;  followed  by  decolorizing  the  sugary 
liquid  with  bone  "coal"  or  vegetable  decolorizing  carbon  and  concen- 
trating under  a  high  vacuum.  The  use  of  starch  with  the  barley  malt 
reduces  the  amount  of  protein  in  the  syrup  and  renders  the  liquid  more 
easily  decolorized  than  straight  malt  syrup. 

Malt  syrup  containing  in  concentrated  form  an  extract  of  hops  as 
well  as  the  maltose  and  other  compounds  from  malt  has  been  produced 
for  small  scale  beer-making  purposes  or  for  export  for  beer  making. 
It  is  made  by  mashing  barley  malt  in  the  usual  manner  as  for  brewing, 
followed  by  boiling  with  hops  and  concentration  in  vacuo  to  a  heavy 
syrup. 

Partially  refined  malt  syrup  diluted  to  about  70°  Brix  makes  a 
satisfactory  table  syrup. 

TOBACCO* 

The  curing  of  tobacco  results  in  the  evolution  of  considerable  heat 
and  rather  profound  changes  in  the  composition,  appearance,  aroma, 
and  flavor  of  the  product.  It  was  thought  at  one  time  that  most  of  the 
changes  were  bacteriological  but  it  now  appears  that  enzymatic  activi- 
ties of  the  plant  are  also  responsible  for  the  transformations  noted. 
From  12  to  40  per  cent,  of  the  dry  matter  of  the  leaves  is  lost  in  the 
various  curing  processes.  The  principal  changes  are  the  disappearance 
of  starch  and  reducing  sugar;  a  decrease  in  pro  terns,  nicotine,  pentosans, 
and  malic  acid;  and  an  increase  in  citric  acid.  Ammonia  is  formed. 
After  drying,  the  leaves  are  piled  in  masses,  moistened,  and  allowed  to 
undergo  a  fermentation  which  raises  the  temperature  to  50°  to  55°. 
In  some  cases  the  leaves  are  sprinkled  with  a  solution  containing  sugar, 

*  Prepared  by  W.  V.  Cruess. 


MICROBIOLOGY   OF   FERMENTED   FOODS  579 

honey,  various  aromatic  substances,  and  sometimes  alcohol  and  passed 
through  another  fermentation. 

The  leaves  are  then  usually  tied  up  in  bundles,  partially  dried, 
pressed  in  boxes  where  a  final  slow  fermentation  occurs.  During  this 
final  fermentation  nicotine  decreases  and  nitrates  are  destroyed,  while 
ammonia  and  sometimes  butyric  acid  are  formed. 

It  seems  probable  that  the  changes  during  the  curing  of  tobacco 
are  due  in  the  first  place  to  hydrolyzing,  proteolytic,  and  oxidizing 
enzymes,  and  that  these  enzymatic  changes  are  supplemented  by  the 
bacteria  which  destroy  nitrates  and  produce  ammonia.  It  is  possible 
that  these  various  factors  account  for  variations  in  the  characteristics 
of  tobacco  from  the  same  source. 

STARCH* 

Starch  is  prepared  from  potatoes,  corn,  wheat,  flour  and  other 
amylaceous  substances.  The  present  method  of  separation  is  by  chem- 
ical means.  Formerly  it  was  accomplished  by  a  complex  fermentation. 

For  the  fermentation  method,  the  grain  is  soaked  in  water  until  soft, 
then  ground  and  made  into  a  paste  which  is  allowed  to  ferment  spon- 
taneously or  started  with  a  leaven  taken  from  a  previous  fermentation. 
Alcoholic,  lactic  and  butyric  microorganisms  attack  the  sugar  while 
others  attack  the  gluten  and  cellulose.  The  fermentation  lasts  from 
twelve  to  twenty-five  days  according  to  the  temperature  and  the 
resistance  of  the  raw  materials. 

During  fermentation,  lactic  and  butyric  acid,  hydrogen  sulphide, 
ammonia  and  carbon  dioxide  with  traces  of  alcohol  and  acetic  acid  are 
produced.  The  process  is  stopped  as  soon  as  gas  ceases  to  be  given 
off  and  before  putrid  fermentation  sets  in.  The  starch  which  is  set 
free  settles  to  the  bottom  and  is  separated  by  decantation,  washing 
and  screening. 

The  washed  starch  is  then  allowed  to  settle  for  three  or  four  days 
in  water.  The  sediment  that  is  formed  consists  of  three  layers,  the  top 
consisting  principally  of  gluten,  the  second  of  gluten  and  starch  and 
the  bottom  of  comparatively  pure  starch.  The  layers  are  separated 
and  the  starch  extracted  from  the  two  upper  layers  by  repeated  wash- 
ings on  inclined  planes.  The  starch,  owing  to  its  higher  specific 
gravity,  remains  near  the  lower  parts  of  these  planes. 

*  Prepared  by  F.  T.  Bioletti. 


580  MICROBIOLOGY  OF  FOODS 

SUGAR* 

In  the  manufacture  of  sugar,  microorganisms  have  no  useful  part 
but  many  forms  may  be  injurious  and  cause  serious  losses.  The 
juices  of  beets  and  sugar  cane  and  the  saccharine  liquids  obtained  by 
presses  or  diffusion  batteries  form  excellent  media  for  the  multiplication 
of  many  Saccharomyces  and  bacteria.  They  are  controlled  by  cleanli- 
ness, rapidity  of  handling,  and  sterilization  by  heat.  They  are  injuri- 
ous by  destroying  sugar  and  thereby  diminishing  the  yield,  by  inverting 
a  portion  of  the  saccharose  and  rendering  the  crystallization  difficult 
and  by  forming  gelatinous  masses  in  the  liquids. 

Many  of  them  are  very  resistant  to  heat.  S.  zopfi  withstands  a 
temperature  of  66°  for  half  an  hour.  Streptococcus  mesenterioides 
forms  chains  of  cocci  surrounded  by  voluminous  gelatinous  sheaths 
which  unite  in  zoogleic  aggregations  sometimes  very  troublesome  in 
sugar  factories.  On  account  of  its  sheath  it  is  very  resistant  to  adverse 
conditions.  It  retains  its  vitality  after  drying  for  three  and  a  half 
years.  It  is  not  killed  by  heating  to  86°  for  five  minutes  and  occurs 
in  the  hot  liquids  of  the  diffusion  batteries. 

TEA* 

In  the  curing  of  black  tea  several  fermentation  processes  occur. 
It  is  stated  that  yeasts  and  bacteria  are  of  little  importance  in  this 
curing  process  under  normal  conditions.  The  most  important  changes 
are  brought  about  by  enzymes  and  by  a  mold  which  converts  the  tan- 
nic  acid  of  the  tea  to  gallic  acid.  If  the  fermentation  is  too  prolonged 
or  moisture  conditions  improper, ^slime-forming  bacteria  develop, 
resulting  in  injury  to  or  complete  spoiling  of  the  product. 

*  Prepared  by  F.  T.  Bioletti. 


CHAPTER  VI* 
MICROBIAL  FOOD  POISONING 

GENERAL  CONSIDERATIONS 

Illness  following  the  ingestion  of  food,  more  or  less  definitely 
ascribable  to  the  food,  has  been  long  recognized.  The  Mosaic  regula- 
tions in  regard  to  foods  forbidden  to  the  Jews  are  evidently  designed  in 
part  to  avoid  the  occurrence  of  food  poisoning.  In  recent  times  recog- 
nized instances  of  food  poisoning  have  been  sufficiently  frequent  to 
make  the  subject  one  of  considerable  importance,  but  there  are  un- 
doubtedly many  instances  of  actual  food  poisoning  in  which  the  causal 
relation  of  the  food  remains  unrecognized  or  even  unsuspected.. 

Food  poisoning  is  usually  suspected  at  once  upon  the  occurrence  of 
sudden  acute  illness  in  a  number  of  people  at  the  same  time,  after  they 
have  partaken  in  common  of  some  particular  food  or  foods.  The 
causal  relation  is  especially  evident  when,  as  sometimes  happens,  a 
large  number  of  people  are  affected  in  the  same  way  immediately  after 
eating  together  at  a  banquet,  not  having  been  associated  with  each 
other  either  before  or  after  the  meal.  When  a  smaller  number  of  indi- 
viduals is  involved,  the  connection  with  food  may  be  more  obscure. 
For  this  reason  most  of  the  well-authenticated  instances  of  food  poison- 
ing are  instances  in  which  many  persons  have  been  affected  at  the  same 
time.  Acute  food  poisonings  involving  only  a  few  persons  probably 
occur  very  frequently  in  the  home,  but  they  receive  little  public  notice 
unless  fatal,  and  are  often  dismissed  as  mere  "errors  in  diet,"  or  as 
"indigestion."  A  careful  study  of  these  cases  is  likely  to  be  made 
only  where  there  is  suspicion  of  criminal  poisoning,  or  some  other 
practical  end  to  be  served  by  the  investigation.  Chronic  forms  of 
food  poisoning  are  for  obvious  reasons  very  difficult  to  recognize  with 
certainty  and  some  of  the  forms  of  disease  the  causation  of  which  has 
been  ascribed  to  chronic  food  poisoning  may  eventually  prove  to  be 
due  to  other  causes.  The  subject  is  still  in  a  very  doubtful  state. 

*  Prepared  by  W.  J.  MacNeal. 


582  MICROBIOLOGY  OP' FOODS 

Several  different  classes  of  food  poisonings  may  be  recognized 
according  to  the  source  of  the  poisonous  substance. 

The  material  of  plants  or  animals  may  be  naturally  poisonous  to 
man  as  a  result  of  the  physiological  activity  of  their  own  living  sub- 
stance. Poison  of  this  kind  may  be  constantly  present  throughout 
the  tissues,  or  it  may  be  confined  to  certain  parts,  or  it  may  occur  only 
at  particular  times  or  seasons.  Some  instances  of  poisoning  with  fish 
and  with  mushrooms  belong  to  this  class,  and  possibly  also  some  of  the 
instances  of  poisoning  with  potatoes  of  high  solanin  content. 

Plants  and  animals  may  feed  upon  substances  not  poisonous  to 
themselves,  and  these  substances  may  remain  a  constituent  part  of 
their  bodies  to  poison  man  when  consumed  by  him.  Some  poisonings 
with  freshly  killed  game  are  considered  to  be  of  this  nature. 

Any  food  may  contain  foreign  poison  added  to  it  by  design  or  by 
accident,  such  for  example  as  the  salts  of  the  various  poisonous  metals. 
The  amount  of  tin  or  lead  passing  into  solution  in  canned  or  tinned 
foods  may  conceivably  be  sufficient  to  cause  poisoning,  but  there  is  no 
reliable  evidence  that  it  has  ever  occurred. 

Animals  may  be  infected  with  pathogenic  bacteria  or  with  other 
parasites  capable  of  infecting  man,  and  the  use  of  food  products  from 
such  animals  may  cause  disease.  Tuberculosis,  trichinosis  and 
tapeworm  may  be  acquired  in  this  way.  . 

Any  food  may  serve  as  the  passive  carrier  of  infectious  agents,  such 
as  B.  typhosus,  and  some  foods  may  even  favor  the  multiplication  of 
pathogenic  bacteria  gaining  access  to  them. 

A  food  may  undergo  chemical  changes  due  to  microorganisms  in- 
capable of  infecting  man,  resulting  in  the  production  of  poisonous  sub- 
stances in  the  food.  Undoubtedly  the  great  majority  of  instances  of 
food  poisonings  belong  in  this  class.  The  bacteria  causing  these  changes 
have  been  designated  as  pathogenic  saprophytes. 

The  last  three  classes  comprise  the  microbial  food  poisonings,  and 
these  are  the  kinds  of  food  poisoning  with  which  we  are  at  present  more 
particularly  concerned. 

INFECTIONS  OF  FOOD-PRODUCING  ANIMALS  TRANSMISSIBLE  TO  MAN 

Animals  dead  of  infectious  diseases  or  slaughtered  in  the  last  stages 
of  disease  are  not  ordinarily  used  for  food,  nor  is  the  milk  of  such 


MICROBIAL   FOOD   POISONING  583 

animals  ordinarily  considered  wholesome.  This  custom  is  certainly 
an  ancient  one,  and  is  doubtless  founded  upon  observation  of  un- 
favorable results  following  the  consumption  of  such  food.  Exact 
knowledge  of  the  nature  of  the  diseases  transmitted  in  this  way  is  a 
more  modern  development,  and  this  more  exact  knowledge  is  now 
being  applied  to  some  extent  through  food-inspection  regulations  to 
prevent  the  transmission  of  such  diseases. 

Tuberculosis  of  cattle  has  been  shown  by  Smith  to  be  due  to  a  germ 
somewhat  different  from  that  causing  the  ordinary  human  tuberculosis, 
and  this  discovery  has  called  into  question  the  necessity  of  avoiding 
the  use  of  food  products  from  tuberculous  animals.  After  a  con- 
siderable amount  of  controversy  it  may  now  be  regarded  as  definitely 
established  that  the  bovine  type  of  tubercle  bacillus  is  capable  of 
infecting  man,  and  that  a  very  considerable  proportion  of  cases  of 
tuberculosis  in  children  is  due  to  this  type  of  organism,  the  infection 
probably  arising  through  the  use  of  milk  from  tuberculous  animals. 
Anthrax,  glanders,  actinomycosis  and  acute  enteritis  of  animals  are 
transmissible  to  man.  Food  products  from  animals  afflicted  with 
these  diseases  should  not  be  used  until  they  have  been  passed  upon  by 
competent  authority.  Further  information  concerning  them  will  be 
found  in  the  sections  dealing  with  these  particular  diseases. 

The  human  disease  known  as  septic  sore  throat  may  be  due  to  in- 
fection with  streptococci  present  in  cow's  milk.  Careful  investigations 
by  various  independent  workers  have  shown  that  these  virulent  strep- 
tococci may  be  derived  from  infected  udders  of  the  cows  and  the  same 
studies  indicate  that  the  infection  in  the  cow  may  be  derived  primarily 
from  human  sources.  In  some  rare  instances  the  disease  in  the  cow 
has  been  traced  to  the  introduction  of  a  milking  tube  into  the  teat 
canal  to  facilitate  the  flow  of  milk  and  the  evidence  against  the  practice 
is  sufficient  to  warrant  its  prohibition. 

In  this  connection  it  may  be  mentioned  that  some  of  the  animal 
parasites,  especially  trichinae  and  various  sorts  of  tapeworms,  gain 
access  to  the  human  body  with  the  food.  Thorough  cooking  usually 
serves  to  kill  these  parasites,  as  well  as  the  pathogenic  bacteria,  but 
ordinary  cooking  should  not  be  too  implicitly  relied  upon  to  accomplish 
this  result. 


584  MICROBIOLOGY   OF.  FOODS 

HUMAN  INFECTIONS  TRANSMITTED  IN  FOOD 

Food  may  serve  as  the  passive  carrier  of  the  germs  of  any  human  in- 
fectious disease  capable  of  indirect  transmission  upon  dead  material. 
In  some  foods,  especially  milk,  these  infectious  agents  may  actually 
multiply.  Typhoid  fever,  diphtheria  and  scarlet  fever  appear  to  be 
rather  frequently  disseminated  through  the  agency  of  food,  and  para- 
typhoid fever  seems  to  be  commonly  transmitted  in  this  way.  Especial 
precautions  are  advisable  to  prevent  persons  afflicted  with  dangerously 
communicable  diseases  and  those  who  are  chronic  germ-carriers  from 
engaging  or  continuing  in  occupations  concerned  with  the  immediate 
preparation  of  food  for  consumption,  particularly  such  occupations  as 
milk  production  and  handling,  market-dairying,  cooking  and  serving 
food.  Numerous  serious  epidemics  have  been  traced  to  such  sources 
in  recent  years.  The  history  of  Mary  Mallon  (Typhoid  Mary)  *  has 
become  popular  knowledge  but  instances  of  similar  spread  of  infection 
are  but  too  common. 

FOOD  POISONING  DUE  TO  THE  GROWTH  OF  SAPROPHYTIC  BACTERIA  IN 

THE  FOOD 

Most  food  poisonings  are  due  to  food  derived  from  perfectly  healthy 
and  wholesome  animals  or  plants,  which  has  subsequently  undergone 
some  bacterial  decomposition  giving  rise  to  poisonous  products.  Our 
knowledge  of  the  specific  causes  of  the  poisonous  changes  is,  however, 
very  incomplete,  and  on  account  of  the  difficult  nature  of  investigation 
in  this  field,  some  of  the  conclusions  reached  by  careful  men  are  still 
open  to  question.  The  bacteria  which  have  been  most  frequently 
identified  with  various  epidemics  of  food  poisoning  are  the  following: 
B.  enteritidis  in  meat  poisoning;  B.  botulinus  in  meat,  sausage  and 
vegetable  poisoning;  B.  paratyphosus  in  poisoning  with  meat,  chicken, 
shellfish,  and  vegetables;  B.  coli  in  cheese  poisoning  and  in  milk  poison- 
ing; B.  vulgaris  in  meat  and  in  vegetable  food  poisonings.  Doubtless 
other  microorganisms,  as  yet  unrecognized,  play  an  important  part  in 
many  food  poisonings,  and  there  is  reason  to  believe  that  some  of  these 
important  unknown  forms  are  anaerobic  bacteria. 

POISONOUS  MEAT  AND  SAUSAGE. — The  flesh  of  a  healthy  animal  is 
ordinarily  free  from  bacteria  at  the  time  of  slaughter,  and  bacterial 

*  Soper,  George  A.:  Typhoid  Mary.     The  Military  Surgeon,  July,  1919,  Vol.  45,  pages  1-15. 


MICROBIAL   FOOD   POISONING  585 

changes  must  begin  at  the  surfaces  of  the  pieces  of  meat  and  gradually 
extend  inward.  In  diseased  animals,  bacteria  more  frequently  circulate 
in  the  blood,  and  the  flesh  may  be  contaminated  throughout  when  the 
animal  dies  of  the  disease  or  when  it  is  slaughtered,  not  only  with  the 
specific  germs  of  the  disease  but  also  with  bacteria  derived  from  the 
intestinal  tract  of  the  animal.  It  is  a  matter  of  observation  that 
the  flesh  of  diseased  animals  is  more  liable  to  undergo  early  putrefac- 
tive and  poisonous  changes  than  that  derived  from  healthy  animals. 
Hashed  meat  is,  of  course,  much  more  prone  to  bacterial  decomposition, 
because  in  it  the  bacteria  have  become  well  distributed  throughout 
the  mass,  and  ideal  conditions  are  provided  for  the  development  of 
anaerobic  as  well  as  aerobic  bacteria.  Minced  chicken  and  chicken 
pie  appear  to  be  very  frequent  sources  of  acute  poisoning  in  the  United 
States,  and  epidemics  of  sausage  poisoning  have  repeatedly  occurred, 
especially  in  Germany.  The  bacteria  found  to  be  concerned  in  these 
instances  have  been  B.  enteritidis,  B.  paratyphosus,  B.  coli,  and  B. 
botulinus.  Some  of  these  poisons,  as  for  example  the  toxin  of  B. 
botulinus,  are  rendered  inert  by  boiling,  but  occasionally  bacterial 
poisons  which  are  not  destroyed  by  such  high  temperatures  may  be 
present  in  food.  *  Moreover,  meat  rendered  poisonous  by  these  bacteria 
may  show  no  evidence  of  putrefaction.  B.  (Proteus)  vulgaris  has 
also  been  found  in  some  samples  of  poisonous  meat,  and  this  finding  is 
usually  associated  with  definite  evidence  of  putrefaction. 

The  symptoms  of  meat  poisoning  are  usually  those  of  acute  gastro- 
enteritis,-— vomiting,  cramps,  and  diarrhoea.  The  patients  often  recover 
very  quickly,  but  occasionally  the  illness  is  rapidly  fatal,  or  it  may 
merge  into  a  subacute  form  resembling  or  identical  with  paratyphoid 
fever.  In  those  instances  of  poisoning  due  to  the  presence  of  B. 
botulinus  the  symptoms  are  of  a  different  kind,  consisting  almost 
solely  of  nervous  disturbances,  secretory  and  motor  paralyses,  without 
fever,  resembling  in  many  respects  poisoning  with  atropin.  In  this 
form  of  meat  poisoning  the  death  rate  is  relatively  high,  about  40  per 
cent  of  the  cases  ending  fatally. 

FISH  POISONING  is  of  two  general  kinds,  that  due  to  poisons  natural 
to  the  fish,  and  that  due  to  poisons  formed  by  bacterial  activity  in  the 
flesh  of  the  fish.  Blanchard  has  applied  the  Spanish  name  "  Siguatera" 
to  the  first  kind  and  the  term  "Botulism"  to  the  second.  In  the 

*  Smith  and  Ten  Broeck.     Jour.  Med.  Research,  1915,  31,  pages  523—546 


586  MICROBIOLOGY   OF  FOODS 

Japanese  fish  of  the  genus  Tetrodon  the  roe  is  poisonous,  giving  rise 
to  severe  gastro-intestinal  irritation  and  convulsions.  The  remainder 
of  the  fish  is  not  poisonous.  In  some  other  fishes  the  sexual  glands 
are  poisonous  during  the  spawning  season;  others  are  provided  with 
special  poison  glands  connected  with  protective  spines  or  barbs. 
These  are  examples  of  poisons  natural  to  fish.  Bacterial  poisons  are 
likely  to  be  formed  in  any  kind  of  fish,  given  the  suitable  conditions, 
and  thus  give  rise  to  the  kind  of  fish  poisoning  designated  as  botulism. 
Cases  of  this  kind  have  resulted  from  eating  (spoiled)  canned  salmon 
and  sardines.  Poisoning  may  also  result  from  eating  diseased  fish, 
the  effects  being  due  to  poisons  elaborated  by  the  infecting  bacteria 
in  the  body  of  the  fish  before  consumption.  This  appears  to  be  a 
rather  common  form  of  fish  poisoning  in  Russia.  B.  paratyphosus  has 
been  isolated  from  some  poisonous  fish,  and  certain  toxicogenic  an- 
aerobes have  been  found  in  others. 

POISONING  WITH  SHELL-FISH  is  so  well  recognized  that  this  form  of 
food  is  not  customarily  used  at  all  during  the  warmer  part  of  the  year, 
May  to  August  inclusive,  the  months  without  an  r  in  their  names. 
Shell-fish  may  serve  as  carriers  of  human  infectious  diseases,  such  as 
typhoid  fever;  they  may  be  poisonous  on  account  of  actual  disease  or 
through  serious  contamination  due  to  living  in  dirty  water;  or  they 
may  be  poisonous  because  of  decomposition  which  has  taken  place 
after  removal  from  the  water.  According  to  the  symptoms  produced, 
there  appear  to  be  at  least  three  distinct  varieties  of  shell-fish  poi- 
soning, one  a  purely  gastro-intestinal  disorder,  the  second  an  involve- 
ment of  the  nervous  system  with  itching  skin  eruption  and  convulsions, 
and  a  third  type  resembling  very  closely  alcoholic  intoxication.  The 
exact  nature  of  the  microbic  agents  concerned  in  these  different  types 
of  poisoning  is  unknown.  It  is  pretty  well  established,  however,  that 
the  poisonous  character  of  shell-fish  is  due  either  to  their  living  for 
some  time  in  dirty  water,  or  to  their  too  long  preservation,  especially 
at  high  temperature,  after  removal  from  the  water. 

MILK,  ICE-CREAM  AND  CHEESE  sometimes  give  rise  to  poisoning,  and 
although  these  instances  are  small  in  number  in  comparison  with  the 
enormous  amount  of  milk  and  milk  products  consumed,  yet  in  the  aggre- 
gate they  are  numerous.  That  many  human  infections  may  be  trans- 
mitted by  milk  has  already  been  pointed  out.  In  summer,  milk  is 
undoubtedly  a  great  factor  in  the  infant  morbidity  and  mortality,  and 


MICROBIAL  FOOD   POISONING  587 

this  poisonous  action  is  largely  due  to  bacterial  changes  in  the  milk. 
Extraordinary  precautions  are  therefore  essential  in  the  production  and 
care  of  milk  to  be  used  as  food  for  children,  particularly  during  the 
warmer  season  of  the  year.  Severe  poisoning  of  adults  with  milk,  ice- 
cream, or  cheese,  is  relatively  less  frequent.  Cases  which  have  been 
studied  have  been  traced  to  the  development  of  B.  coll  or  B.  paraty- 
phosus  in  these  foods.  There  is  some  evidence  that  other  bacteria, 
probably  strict  anaerobes,  are  also  sometimes  concerned.  Strict 
cleanliness,  proper  refrigeration,  and  pasteurization  of  milk  of  uncertain 
character  may  usually  be  relied  upon  to  prevent  milk  poisoning. 
Ice-cream  should  be  made  only  from  wholesome  materials  and  with 
due  regard  to  cleanliness  in  making  it.  The  causes  of  serious  cheese 
poisoning  are  not  definitely  known,  but  such  poisoning  may  be  avoided, 
to  a  large  extent  at  least,  by  using  only  standard  varieties  of  cheese  of 
the  proper  odor  and  flavor. 

VEGETABLE  FOOD  POISONING,  in  an  acute  form,  has  followed  the  use 
of  sprouting  and  partly  decomposed  potatoes,  and  also  various  canned 
vegetables,  particularly  those  of  high  protein  content,  such  as  beans. 
The  large  majority  and  possibly  all  of  these  cases  are  due  to  decomposi- 
tion changes  in  the  foods,  B.  botulinus  and  B.  proteus  appearing  to  be 
the  microbes  most  frequently  concerned. 

SPECIFIC  DISEASES  DUE  TO  FOOD  POISONING 
BOTULISM  AND  BACILLUS  BOTULINUS. — Perhaps  the  most  serious 
and  most  rapidly  fatal  of  all  the  food  poisonings  is  botulism,  a  disorder 
caused  by  a  true  bacterial  toxin  formed  in  food  previous  to  its  ingestion, 
by  the  growth  of  a  specific  anaerobic  organism,  Bacillus  botulinus. 
The  earliest  recognized  cases  of  this  disease  were  observed  in  Wiirtem- 
berg,  Germany,  and  followed  the  eating  of  sausages,  hence  the  name 
botulism  or  sausage  poisoning.  Mayer  has  recorded  812  cases  of  botu- 
lism in  Germany  up  to  1913,  with  365  deaths.  Dickson*  has  collected 
records  of  64  cases  in  the  United  States  from  1894  to  1918,  of  which  41 
resulted  in  death.  The  mortality  in  this  series  has  been,  therefore, 
64  per  cent.  It  seems  certain  that  only  a  small  proportion  of  actual 
existing  cases  has  been  recognized  and  that  milder  outbreaks  of  the 
poisoning,  especially,  have  escaped  record.  In  one  outbreak  the 
mortality  was  100  per  cent,  and  ;n  another  only  8.3  per  cent. 

*  Dickson,  E.  C.:  Botulism.  Monograph  No.  8  of  the  Rockefeller  Institute  for  Medical 
Research,  1918,  p.  51. 


588  MICROBIOLOGY   OF   FOODS 

The  first  symptoms  of  botulism  appear,  as  a  rule,  in  from  eight  to 
one  hundred  and  twenty  hours  after  the  poisonous  food  has  been  taken. 
In  exceptional  instances  they  may  appear  as  early  as  two  hours  after 
the  meal  or  be  delayed  until  the  ninth  or  tenth  day.  In  most  instances 
the  first  symptoms  are  referable  to  paralysis  of  motor  or  secretory  nerves. 
Double  vision  due  to  paralysis  of  the  external  recti  or  even  of  all  the 
extra-ocular  muscles,  dilated  and  sluggish  pupils,  difficulty  in  swallow- 
ing, dryness  of  the  mouth,  nausea  and  vomiting,  accumulation  of 
mucus  in  the  paralyzed  pharynx  resulting  in  paroxysms  of  choking, 
persistent  constipation,  rapidly  progressive  weakness,  with  undis- 
turbed sensation  and  clear  mentality,  are  the  most  characteristic 
manifestations  of  the  disease.  Disturbance  of  vision  is  often  the  initial 
symptom.  Less  frequently  the  illness  begins  with  vomiting.  Usu- 
ally the  temperature  remains  subnormal  but  fever  is  present  in  some 
instances,  possibly  on  account  of  complicating  bronchopneumonia  or 
other  terminal  infection.  The  following  graphic  description  of  the 
fully  developed  disease  is  quoted  from  Dickson's  monograph  (page  46) : 
"The  general  appearance  of  the  patient  is  most  distressing.  The  ex- 
treme muscular  weakness,  the  anxiety  and  the  utter  helplessness,  the 
difficulty  in  swallowing,  the  attacks  of  strangling,  the  struggle  for 
breath,  and  the  unsuccessful  attempts  to  articulate  constitute  a  clinical 
picture  which,  when  once  observed,  can  never  be  forgotten.  The 
face  is  usually  pale,  but. in  the  early  stages  may  be  congested.  There 
may  be  normal  appetite  and  excessive  thirst,  but  the  patient  is  afraid 
to  try  to  swallow.  At  times  the  strangling  spells  are  so  severe  that 
there  is  incontinence  of  urine,  and  the  accumulation  of  thick,  tenacious 
mucus  in  the  pharynx  is  a  constant  source  of  annoyance.  The  fact 
that  the  patient  remains  in  full  possession  of  his  mental  powers  and 
can  realize  the  seriousness  of  his  condition  only  adds  to  the  distressing 
character  of  the  situation." 

At  autopsy,  congestion  of  the  central  nervous  system  is  constantly 
found  and,  in  nearly  every  case,  thrombosis  of  the  meningeal  vessels 
as  well  as  the  blood  vessels  in  various  other  organs.  The  ganglion 
cells  are  well  preserved  in  many  cases  although  some  of  the  earlier 
observers  recorded  disturbances  of  the  Nissl  granules  and  displacement 
and  distortion  of  the  nucleus  of  the  motor  cells.  The  cerebrospinal 
fluid  contained  80  cells  per  cu.  mm.  in  one  case  during  life.  The 
differential  diagnosis  of  a  single  case  of  the  disease  in  the  absence  of 


MICROBIAL    FOOD    POISONING  589 

history  suggesting  food  poisoning  presents  great  difficulties  and  there 
is  good  reason  to  believe  that  many  instances  of  botulism  fail  to  be 
recognized  during  life  or  at  autopsy. 

Various  animals  are  subject  to  botulism.  It  has  been  established 
that  horses,  mules  and  domestic  fowl  suffer  and  die  from  the  disease 
naturally.  Dogs,  cats,  guinea-pigs  and  rabbits  are  susceptible  to  the 
experimental  disease.  Cattle  and  chickens,  though  susceptible,  seem 
distinctly  more  resistant  than  horses.  Graham  and  his  associates  have 
brought  forward  convincing  evidence  to  prove  that  at  least  some  ex- 
amples of  "Forage  poisoning"  and  "Ensilage  poisoning"  in  domestic 
animals  are  actually  due  to  toxin  of  B.  botulinus  produced  in  the  feed. 
In  these  animals  paralysis  and  muscular  weakness  are  the  prominent 
manifestations.  The  disease  has  been  designated  as  limber  neck  in 
chickens  and  as  cerebro-spinal  meningitis,  staggers  or  blind  staggers  in 
horses.  The  proof  that  the  disease  is  botulism  rests  upon  the  isola- 
tion of  B.  botulinus  from  the  food  which  gave  rise  to  the  poisoning, 
or  the  effective  protection  of  experimental  animals  against  the  poison 
in  the  food  by  administration  of  specific  botulinus  antitoxin  to  them 
while  unprotected  control  animals  are  fatally  poisoned,  or  by  the 
successful  results  of  both  these  experimental  procedures. 

Bacillus  botulinus  is  a  large  rod  0.9  to  i.2/i  wide  and  4  to  6/x  long, 
single,  in  pairs  or  in  longer  threads  when  growth  conditions  are  unfav- 
orable. The  spore  is  oval  and  causes  enlargement  of  the  cell.  It  is 
usually  terminal  or  near  one  end,  but  may  be  central.  The  bacillus 
is  slightly  motile,  possesses  4  to  8  flagella  and  is  Gram-positive.  It  is 
a  strict  anaerobe,  although,  like  other  anaerobes,  capable  of  active 
growth  in  symbiosis  with  aerobic  bacteria  in  the  presence  of  air.  Glu- 
cose and  salt  in  dilute  solution  favor  growth  but  a  concentration  of 
6  per  cent,  sodium  chloride  inhibits  development.  The  optimum  tem- 
perature for  growth  is  about  28°C.  Below  16°  and  above  37°C.  growth 
is  slight.  The  spores,  according  to  Van  Ermengem,  are  killed  in  a 
half  hour  at  8o°C.  and  by  boiling  for  five  minutes.  More  recent  care- 
ful tests  by  Burke  have  shown  that  this  earlier  work  cannot  be  relied 
upon  and  that  there  is  considerable  variability  in  the  resistance  of  the 
spores  produced  on  different  culture  media.  She  found  that  some  of  the 
spores  resist  boiling  water  for  two  hours  and  the  spores  of  one  strain 
resisted  heat  in  the  autoclave  at  5  pounds  pressure  for  ten  minutes. 
The  importance  of  these  observations  in  relation  to  canned  food  is 
obvious. 


5QO  MICROBIOLOGY   OF   FOODS 

The  poison  of  B.  botulinus  is  a  true  bacterial  toxin,  which  in  its 
potency  belongs  in  a  class  with  the  toxin  of  diphtheria  and  tetanus. 
Dickson  has  produced  a  crude  toxin  of  which  o.oooi  c.c.  killed  a  guinea- 
pig  within  twenty-four  hours.  Unlike  these  other  toxins,  however, 
botulin  (the  botulinus  toxin)  is  actively  poisonous  when  swallowed 
with  food  as  well  as  when  injected  into  the  tissues.  An,  antitoxic 
serum  has  been  produced  by  immunization  of  goats.  This  serum  has 
considerable  value  in  preventing  the  disease  but  little  value  in  treat- 
ment after  the  symptoms  have  appeared.  The  analogy  with  tetanus 
is  evident.  Botulin,  like  the  tetanus  toxin,  has  a  strong  avidity  for 
nerve  tissue.  The  toxin  loses  strength  slowly  when  heated  at  56°C. 
and  is  rendered  harmless  by  heating  at  8o°C.  for  thirty  minutes  or  by 
boiling  for  five  minutes. 

Botulism  has  long  been  known  as  a  form  of  meat  poisoning  and  it 
has  also  been  known  that  vegetable  foods  of  high  protein  content,  such 
as  beans,  might  give  rise  to  this  poisoning.  The  clinical  and  experi- 
mental observations  of  Dickson,  Graham  and  his  associates  have  called 
attention  to  the  possible  production  of  botulinus  toxin  in  various  vege- 
table foods,  including  canned  corn,  asparagus,  spinach,  apricots  and 
peaches  as  well  as  oats,  hay  and  ensilage.  The  virulence  of  the  poison 
is  such  that  a  mere  taste  of  the  tainted  food  is  sufficient  to  cause  serious 
illness  and  the  swallowing  of  a  single  spoonful  has  caused  fatal  poisoning. 

Botulism  may  be  caused,  therefore,  not  only  by  the  consumption 
of  meats  and  meat  products  but  also  of  vegetable  foods  and  it  is  especially 
important  at  this  time  to  emphasize  the  danger  in  canned  foods,  espe- 
cially in  home  canned  foods.  So  far,  botulism  has  been  traced  to  com- 
mercial canned  foods  very  rarely  indeed  but  the  methods  used  at 
home,  especially  the  cold-pack  method,  may  be  quite  inadequate  to 
destroy  spores  of  B.  botulinus  if  they  have  gained  access  to  the  food.  The 
canning  of  vegetables  which  are  not  sound  and  clean  is  an  important 
source  of  danger.  Even  in  commercial  canning  with  standardized  methods 
and  control  it  is  doubtful  whether  the  heating  is  adequate  to  destroy 
spores  of  B.  botulinus.  Weinzirl*  has  found  viable  spores  of  aerobic 
bacteria  in  marketable  commercial  canned  foods  and  he  regards  the 
absence  of  oxygen  as  essential  to  the  preservation  of  such  canned  foods. 
If  the  spores  of  B.  botulinus  are  as  resistant  as  appears  from  the  studies 
of  Burke,  then  we  must  expect  botulism  from  commercial  canned  foods 

*  Weinzirl,  J.     The  bacteriology  of  canned  foods.     Journal  of  Med.  Rsch.,  Jan.,  1919, 
Vol.  39,  No.  3,  P-  348-413. 


MICROBIAL   FOOD    POISONING  59 1 

unless  B.  bolulinus  is  excluded  by  critical  selection  of  sound  materials 
and  cleanliness  throughout  the  canning  process. 

Signs  of  spoilage  due  to  B.  botulinus  may  not  be  evident  at  all. 
Careful  examination  will  often  reveal  gas  bubbles  in  the  container,  an 
odor  suggesting  rancid  cheese  and  a  mushy  disintegrated  appearance 
of  the  solid  contents.  Canned  foods  showing  any  of  these  signs  should 
never  be  tasted  until  they  have  been  cooked  again.  Indeed,  Dickson 
concludes  that  all  home  canned  foods  should  be  cooked  again  before 
being  eaten.  Boiling  for  five  minutes  just  before  serving  the  food 
practically  removes  the  danger  of  botulism. 

Ergotism  is  a  disease  characterized  by  cachexia,  gangrene  and  con- 
vulsions. It  is  caused  by  eating  the  fungus,  Claviceps  purpurea,  which 
grows  as  a  parasite  upon  rye.  The  grain  of  this  parasite  has  a  consid- 
erable commercial  (medicinal)  value  sufficient  to  pay  for  its  separation 
from  rye  where  it  occurs,  so  there  is  little  economic  excuse  for  food 
poisoning  from  this  cause. 

Beriberi  or  kakke  is  an  acute  or  chronic  nervous  disorder  which  has 
been  observed  especially  in  the  Orient,  Japan  and  the  Philippine  Is- 
lands, although  it  has  also  been  found  in  Brazil,  in  Labrador  and  rather 
frequently  •  among  sailors  after  long  sea  voyages.  At  one  time  the 
disease  was  ascribed  to  the  use  of  fish  as  food,  later  to  the  use  of  rice. 
Modern  studies,  especially  those  of  Chamberlain,  Vedder  and  theii 
associates  in  the  Philippine  Islands,  have  shown  that  beriberi  may 
be  prevented  by  including  beans,  unpolished  rice  or  rice  hulls  in  suffi- 
cient quantity  in  the  diet  and  furthermore  that  those  already  afflicted 
with  the  disease  usually  recover  completely  when  given  these  foods 
or  when  treated  with  an  alcoholic  extract  of  rice  polishings.  The 
curative  principle  of  rice  polishings  has  been  studied  by  Funk  who 
has  named  it  vitamine.  He  ascribes  the  causation  of  beriberi  to  a  lack 
of  this  supposedly  necessary  vitamine  in  the  food  and  this  theory  has 
been  very  favorably  received.  It  must  be  acknowledged,  however, 
that  the  etiology  of  beriberi  is  still  not  convincingly  proven.  The 
discovery  of  a  remedy  which  eradicates  a  given  disease  is  not  sufficient 
to  prove  that  the  lack  of  this  particular  therapeutic  agent  is  the  es- 
sential cause  of  the  disease. 

Pellagra  is  a  cachexia,  characterized  by  a  definite  sort  of  skin  erup- 
tion, which  has  been  ascribed  to  the  use  of  maize  (Indian  corn)  as 
food.  This  disease  is  discussed  in  a  separate  section  (page  865). 


592  MICROBIOLOGY  OF  FOODS 

THE  CHEMICAL  NATURE  OF  FOOD  POISONS 

The  poisonous  substances  in  foods  are  for  the  most  part  of  the  same 
nature  as  the  poisons  of  the  pathogenic  bacteria.  The  simplest  in 
structure  of  these  poisons  belong  to  the  alkaloidal  substances,  substi- 
tuted ammonia  and  ammonium  compounds,  called  ptomains  (page  241). 
Several  of  these  have  been  prepared  in  a  pure  state,  for  example,  mytilo- 
toxin  (C6Hi5N02)  from  poisonous  shell-fish  and  neurin  (C2H3-N- 
(CH^aOH)  from  putrefied  horse,  beef,  and  human  flesh.  Although 
ptomains  undoubtedly  occur  at  times  in  poisonous  foods,  they  are  not 
now  considered  of  so  much  importance  in  food  poisoning  as  formerly, 
for  in  the  majority  of  samples  of  poisonous  food  the  search  for  ptomains 
has  been  in  vain.  The  poisonous  effects  are  believed  rather  to  be  due 
for  the  most  part  to  much  more  complex  bodies  resulting  from  the 
earliest  analytic  changes  in  the  food  protein,  or  else  to  bodies  built  up 
by  actual  synthesis  by  the  bacteria.  Such  substances  are  classed  with 
the  toxic  proteins  and  the  true  toxins.  Their  chemical  composition 
and  structure  are  not  definitely  known. 

REFERENCES 

Burke,  Georgina  Spooner,  The  effect  of  heat  on  the  spores  of  B.  botulinus.  Its 
bearing  on  home  canning  methods:  Part  I,  Journ.  A.M.A.,  Jan.  n,  IQIQ,  Vol.  72, 
No.  2,  pp.  88-92. 

Dickson,  Ernest  C.,  Botulism,  A  clinical  and  experimental  study.  Monographs 
of  the  Rockefeller  Institute  for  Medical  Research,  No.  8,  July  31,  1918. 

Dieudeomne,  A.,  translation  by  Bolduan,  C.  F.,  Bacterial  food  poisoning.  E.  B. 
Treat  and  Co.,  New  York,  1909. 

Graham,  Robert  and  Brueckner,  A.  L.,  Studies  in  forage  poisoning.  Journal  of 
Bacteriology,  Jan.,  1919,  Vol.  4,  No.  i,  pp.  1-21.  Graham,  Brueckner  said  Pontius, 
R.  L.,  Studies  in  forage  poisoning.  Kentucky  Agr.  Exp.  Sta.  Bull.,  No.  207-208, 
Lexington,  June  and  July,  1917,  pp.  47-113. 

Novy,  F.  G.,  Food  poisons,  Osler-McCrae,  Modern  Medicine,  1914,  Vol.  II,  pp. 
450-471. 

Smith,  Theobald  and  TenBroeck,  Carl,  The  pathogenic  action  of  the  fowl  typhoid 
bacillus  with  special  reference  to  certain  toxins.  Jour.  Med.  Rsch.,  Jan.,  1915, 
Vol.  31,  No.  3,  p.  523-546. 

Thresh  and  Porter,  Preservatives  in  food  and  food  examination.  J.  and  A. 
Churchill,  London,  1906. 

Vaughan  and  Novy,  Cellular  toxins.    Lea  Bros,  and  Co.,  Philadelphia,  1902. 

Weinzirl,  J.  The  bacteriology  of  canned  foods,  Jour.  Med.  Rsch.,  Jan.,  1919, 
Vol.  39,  No.  3,  p.  348-413. 


CHAPTER  VII* 
MICROORGANISMS  OF  THE  DIGESTIVE  TRACT 

INTRODUCTION 

The  digestive  tube  of  the  vertebrate  animal  is  in  communication 
with  the  external  world  and  is  the  passageway  for  a  great  variety  of 
materials  constituting  the  food  of  the  animal.  This  food  brings  with 
it  various  sorts  of  microbes,  at  times  in  considerable  numbers.  Within 
the  digestive  tube  the  food  is  more  or  less  completely  resolved  by  the 
processes  of  digestion  into  soluble  nutritive  split  products,  which  furnish 
an  excellent  medium  for  microbic  development.  It  is  not  surprising, 
therefore,  that  there  is  an  enormous  multiplication  of  microorganisms 
within  the  intestine,  both  in  health  and  disease,  and  that  this  multi- 
plication is  most  active  during  the  digestion  of  the  food. 

MICROORGANISMS  OF  CERTAIN  PORTIONS  OF  THE  ALIMENTARY  CANAL 

The  entire  digestive  tract  is  free  from  microbes  during  normal 
intrauterine  life.  After  birth  the  canal  is  quickly  invaded  by  bacteria, 
chiefly  through  the  mouth  and  nose,  but  to  a  lesser  extent  also  through 
the  anal  orifice.  In  the  mouth,  pharynx  and  intestine,  some  of  these 
invaders  establish  themselves  to  remain  throughout  the  life  of  the 
individual  host.  The  species  of  microbes  present  and  the  numerical 
proportions  of  the  different  species  of  normal  buccal  and  intestinal 
microorganisms  vary  somewhat  with  the  age  of  the  host  and  the  charac- 
ter of  his  food.  They  are  also  considerably  disturbed  sometimes  by 
the  entrance  and  multiplication  of  pathogenic  germs,  giving  rise  to 
disease  in  their  host,  such  as  Oidium  albicans  in  the  mouth  or  the  chol- 
era vibrio  in  the  intestine. 

MICROORGANISMS  OF  THE  MOUTH. — The  buccal  cavity  presents 
conditions  of  temperature,  moisture,  chemical  reaction  and  a  variety 
of  food  substances  in  its  various  parts,  which  are  very  favorable  to  the 
growth  of  many  microbic  species.  Aerobic,  facultative  and  anaerobic 
forms  are  found  and  the  species  are  very  numerous.  Miller,  in  a  few 

*  Prepared  by  W.  J.  MacNeal. 

38  593 


594  MICROBIOLOGY  OF  FOODS 

weeks,  was  able  to  isolate  more  than  a  hundred  different  kinds  of  bac- 
teria from  the  mouth.  Many  of  these  are  doubtless  only  transient 
residents,  having  gained  entrance  with  food,  water  or  air. 

Among  the  almost  constant  inhabitants  of  the  mouth  may  be 
mentioned  the  streptococci,  both  the  variety  which  produces  a  green 
color  on  bloodagar,  the  Strept.  salivarius  or  Strept.  viridans,  and  the 
hemolytic  variety,  Strept.  hcemolyticus;  the  M.  pyogenes  var.  aureus  and 
albus;  the  lodococcus  magnus  and  lodococcus  parvus  of  Miller,  which 
may  be  cultivated  upon  a  sugar-starch  gelatin-agar  medium  and  are 
stained  blue  by  iodine;  two  or  three  species  of  spirilla,  described  by 
Miller,  which  may  be  cultivated  with  some  difficulty  upon  ordinary 
nutrient  agar;  B.ftisiformis  of  Vincent,  which  may  be  cultivated  as  a 
strict  anaerobe  in  media  containing  blood  serum  or  ascitic  fluid;  B. 
maximus  (buccalis)  of  Miller,  a  bacillus  forming  threads  0.5  to  1.5^. 
wide  and  2o/z  or  more  in  length,  cultivable  upon  maltose  agar  or 
potato  gelatin;  Leptothrix  buccalis,  a  slender  unbranched  filament, 
which  may  be  brought  to  development  on  ordinary  media,  with  some 
difficulty. 

Even  more  definitely  characteristic  mouth  bacteria  are  those 
which  are  found  in  every  human  mouth  (except  in  very  young  children) 
and  which  are  not  cultivable  in  artificial  media  at  all  or  only  under 
special  artificial  conditions  never  met  with  in  nature.  Among  these 
forms  may  be  mentioned  the  lodococcus  vaginatus,  an  encapsulated 
organism  which  may  be  stained  blue  by  Lugol's  solution  acidified  by 
addition  of  lactic  acid;  the  Sp.  sputigenum,  which  is  found  especially  at 
the  inflamed  margin  of  the  gums;  the  Spirochceta  buccalis,  Spirochceta 
media,  Spirochceta  microdentium  and  macro dentium,  organisms  which 
are  found  in  the  mucus  about  the  teeth,  but  are  especially  numerous 
on  denuded  areas  or  in  abscess  cavities  of  the  gums  or  in  carious  teeth. 
The  spirochetes  of  the  mouth  have  been  successfully  cultivated  by 
anaerobic  methods  in  serum  and  in  ascitic  fluid  by  several  investigators, 
notably  by  Noguchi.* 

The  amceba  of  the  mouth,  Entamoeba  (Endanmba)  buccalis,  may  be 
found  in  nearly  every  individual  in  the  deposits  between  the  teeth  and 
especially  in  carious  teeth.  The  cell  is  6  to  32,11  in  diameter,  actively 
motile,  with  few  lobose  pseudopodia.  The  nucleus  of  the  living  amceba 
is  visible.  Its  food  apparently  consists  of  bacteria  and  the  bodies  of 

•  Noguchi,  H.:  Jour.  Exp.  Med.,  1912,  XV,  81. 


MICROORGANISMS    OF   THE   DIGESTIVE   TRACT  595 

leucocytes.  It  does  not  appear  to  penetrate  living  tissue.  Other 
mouth  amoebae  have  been  described.  Whether  they  really  belong  to 
species  distinct  from  Entamosba  buccalis  is  questionable.  Recently  it 
has  been  claimed  that  the  amoebae  of  the  mouth  bear  a  causal  rela- 
tion to  pyorrhea  alveolaris,  but  the  claim  has  not  been  convincingly 
proven. 

The  various  characteristic  buccal  microorganisms  are  found  in 
particular  parts  of  the  mouth  and  their  numbers  vary  considerably 
according  to  the  cleanliness  of  the  mouth  and  teeth,  presence  or  absence 
of  denuded  areas,  ulcers,  sinuses  or  carious  teeth.  The  iodine-staining 
varieties  are  especially  abundant  between  the  teeth  and  upon  starchy 
food  residues.  The  spirochetes,  on  the  other  hand,  are  more  abundant 
in  the  serum  exuding  from  denuded  areas  and  in  pus  cavities.  B. 
fusiformis  (Vincent)  is  often  found  in  normal  buccal  mucus  but  it  is 
especially  abundant  in  the  necrotic  ulcers  of  the  tonsil  in  the  disease 
known  as  Vincent's  angina,  in  which  situation  it  is  always  associated 
with  numerous  spirochetes. 

Some  of  the  members  of  the  normal  mouth  flora  occasionally  play 
definite  pathogenic  roles.  There  can  be  little  doubt  that  the  starch- 
fermenting  forms  produce  acid,  thus  attacking  the  mineral  matter  of 
the  teeth  and  favoring  dental  caries.  The  pathogenic  role  of  Strept. 
viridans,  when  it  penetrates  into  carious  teeth,  causing  root  abscess 
and,  probably  by  metastasis  from  this  focus,  giving  rise  to  arthritis  and 
endocarditis,  is  indicated  by  a  mass  of  circumstantial  and  experimental 
evidence  which  is  well  nigh  convincing.  The  frequently  serious  nature 
of  infections  with  the  hemolytic  streptococcus  are  well  known.  Doubt- 
less members  of  this  variety  of  streptococcus  in  the  mouth  are  ready 
to  acquire  virulence  whenever  lowered  resistance  of  the  host  presents 
a  favorable  opportunity  for  them  to  invade  the  tonsils,  the  pharyngeal 
mucous  membrane,  the  Eustachian  tube  and  middle  ear,  not  to  mention 
more  distant  parts  of  the  body. 

Certain  very  specific  pathogenic  microorganisms  are  found  in  the 
mouth  and  pharynx  from  time  to  time  and  they  sometimes  produce 
lesions  there.  Spirochceta  pallida  is  especially  abundant  in  the  buccal 
and  pharyngeal  lesions  of  secondary  syphilis.  The  tubercle  bacillus 
is  expectorated  through  the  mouth  in  open  pulmonary  tuberculosis. 
The  pneumococcus  is  often  found  in  the  mouth  and  pharynx,  even  in 
health  and  is  especially  numerous  and  virulent  in  cases  of  lobar  pneu- 


5Q6  MICROBIOLOGY   OF   FOODS 

monia.  The  influenza  bacillus  and  Bact.  diphtheria  are  also  occasion- 
ally found  in  the  throats  of  healthy  persons  as  well  as  of  those  suffering 
from  the  diseases  to  which  they  give  rise. 

MICROORGANISMS  OF  THE  STOMACH. — The  microbic  flora  of  the 
healthy  stomach  consists  almost  exclusively  of  organisms  swallowed. 
The  gastric  juice  restrains  bacterial  multiplication  and  kills  a  large 
majority  of  the  bacteria  which  enter  the  stomach.  In  diseased  con- 
ditions the  absence  or  reduced  concentration  of  the  hydrochloric  acid 
may  permit  the  multiplication  of  yeasts,  of  large  lactic-acid  bacilli 
(Boas-Oppler  bacilli),  of  encapsulated  cocci  (Sarcina  ventriculi),  or 
even  of  flagellate  protozoa,  such  as  Lamblia  and  Trichomonas. 

MICROORGANISMS  OF  INTESTINE. — The  duodenum  receives  from  the 
healthy  stomach  relatively  few  living  bacteria.  The  secretions  of 
the  liver,  pancreas  and  of  the  duodenal  wall  are  very  free  from  bacteria 
and  they  tend  to  flush  out  the  duodenum.  In  health  this  portion  of 
the  intestine  is  quite  free*  from  living  bacteria  in  the  intervals  when 
food  is  absent  and  it  contains  relatively  few  bacteria  during  digestion. 
Among  the  living  microorganisms  most  frequently  found  here  are  Gram- 
positive  cocci  which  fail  to  liquefy  gelatin.  B.  coll  is  uncommon.  In 
spite  of  the  negative  results  of  culture  work  upon  duodenal  juice,  it  is 
always  possible  to  see  with  the  microscope  abundant  bacterial  cells  in 
it.  These  are  probably  dead. 

From  the  upper  end  of  the  jejunum  to  the  ileocecal  valve,  the 
number  of  bacteria  in  the  small  intestine  progressively  increases.  In 
the  intervals  when  food  is  absent,  even  these  portions  of  the  small 
intestine  tend  to  free  themselves  from  bacteria,  in  part,  probably, 
because  they  are  continually  flushed  out  by  the  intestinal  secretion, 
but  probably  in  part  also,  as  has  been  maintained  by  Kohlbrugge,f 
because  of  a  definite  bactericidal  property  of  the  intestinal  mucous 
membrane.  However  this  may  be,  it  is  certain  that  living  organisms 
of  the  B.  coll  group  and  various  streptococci  are  commonly  found  in 
intestinal  contents  taken  from  the  jejunum  or  ileum  at  operation  or  at 
autopsy  and  that  these  organisms  are  quite  numerous  in  the  material 
discharged  from  the  lower  end  of  the  small  intestine  in  cases  of  ileo- 
cecal fistula.  J  The  relative  abundance  of  the  different  kinds  of  bacteria 

*  MacNeal  and  Chace,  Arch.  Int.  Med.,  1913,  XII,  178. 

t  Kohlbrugge,  Centrabl.  f.  Bakt.  Abt.  I,  1901,  XXIX,  571;  ibid.,  1901,  XXX,  10;  ibid., 
1901,  XXX,  70. 

t  Macfayden,  Nencki  und  Sieber,  Arch.  f.  Exp.  Path,  und  Pharm.,  1891,  XXXIII,  311. 


MICROORGANISMS    OF   THE   DIGESTIVE   TRACT  597 

may  be  altered  by  changing  the  character  of  the  diet,  a  fact  of  impor- 
tance in  the  treatment  of  intestinal  infections. 

In  the  caecum  there  is  a  sudden  enlargement  of  the  lumen  of  the 
intestinal  canal  and  a  consequent  retardation  of  the  movement  of 
the  intestinal  contents.  The  blind  pouch  also  favors  stagnation.  In 
this  region  the  whole  intestinal  contents  usually  acquire  a  chemical 
reaction  neutral  or  alkaline  to  litmus.  All  these  factors  favor  the 
enormous  multiplication  of  bacteria.  Indeed,  the  caecum  and  the 
remaining  large  intestine  constitute  the  great  bacterial  incubator  of 
the  healthy  body.  Here  B.  coli  multiplies  enormously;  the  strict 
anaerobes,  Bad.  Welchii  and  B.  edematis  flourish  under  most  favorable 
conditions.  Various  streptococci,  staphylococci  and  spirochetes  multi- 
ply either  in  the  food  residues  or  in  the  intestinal  secretions.  An 
easily  digested  mixed  diet  favors  the  facultative  anaerobes,  while  excess- 
ive feeding  of  starchy  foods  and  of  meat  leads  to  an  overgrowth  of  the 
strict  anaerobes,  especially  those  of  the  Bad.  Welchii  group.  Many  of 
these  bacteria  will  then  be  found  to  stain  blue  with  iodine,  giving  the 
so-called  granulose  reaction.  A  milk  diet,  especially  if  limited  in 
amount  and  well  digested  by  the  individual,  favors  the  micro-aerophilic 
B.  bifidus  of  Tissier,  the  organism  which  is  dominant  in  the  faeces  of  the 
healthy  breast-fed  infant  and  occasionally  very  abundant  even  in 
adults. 

In  the  lower  portions  of  the  large  intestine,  as  a  result  of  progressive 
absorption  from  the  contents  of  the  bowel,  there  is  a  concentration 
and  overcrowding  of  the  bacteria  which  have  developed  at  higher  levels. 
The  vast  majority  of  them  die  and  these  dead  cells,  together  with  the 
still  abundant  living  microorganisms,  make  up  about  a  third  of  the 
substance  of  the  faeces.  The  faeces  are  composed  of  rejected  food 
residue?,  residues  of  intestinal  secretions,  of  bile  and  pancreatic  juice 
and  abundant  microorganisms,  some  of  the  latter  still  actively  multi- 
plying, but  the  majority  of  them  dead  and  in  various  stages  of 
disintegration. 

THE  MICROORGANISMS  OF  THE  FAECES. — The  microorganisms  of  the 
faeces  represent  the  end  result  of  the  progressive  multiplication  or  dis- 
integration, or  both,  of  the  organisms  originally  present  in  the  food 
together  with  all  those  added  at  various  regions  of  the  alimentary  canal. 
The  microbic  flora  of  the  large  intestine  is,  however,  most  prominent 
in  the  faeces.  The  total  quantity  and  the  proportions  of  the  various 


598  MICROBIOLOGY  OF  FOODS 

kinds  of  microbes  in  the  faeces  varies  considerably  even  in  health,  depend- 
ing upon  various  factors,  among  which  age  of  the  individual  and 
character  of  the  food  are  very  important. 

The  first  meconium  passed  after  birth  may  contain  few  or  no  micro- 
organisms. Within  a  few  hours,  however,  they  appear  in  the  intestinal 
discharges.  The  earliest  forms  are  usually  large  diplococci  to  which 
are  soon  added  various  bacilli,  small  diplococci  and  tetrads.  Among 
the  bacilli,  a  long  slender  form  with  a  large  oval  terminal  spore,  the 
headlet  bacillus  of  Escherich,  is  particularly  conspicuous.  B.  coli  is 
also  present  at  this  time  and  several  gelatin-liquefying  forms  of  bacilli 
can  be  isolated  in  cultures,  among  them  B.  (Proteus)  vulgaris  and  B, 
subtilis.  Anaerobic  cultures  demonstrate  the  presence  of  Bad.  Wekhii, 
B.  edematis  and  B.  bifidus. 

As  the  meconium  is  replaced  by  the  residue  of  the  ingested  mother's 
milk,  the  previously  variegated  bacterial  flora  suddenly  becomes  very 
simple  and  during  the  whole  period  of  exclusively  breast  feeding  the 
stools  contain  enormous  numbers  of  the  Gram-positive  micro-aerophilic 
B.  bifidus  of  Tissier,  with  only  small  numbers  of  B.  coli  and  very  few  cocci. 
The  dominance  of  B.  bifidus  may  readily  be  demonstrated  by  making  a 
series  of  dilution  cultures  in  tall  tubes  of  glucose  agar,  according  to  the 
method  of  Veillon,  and  incubating  them  for  five  days  or  more.  When 
the  child  begins  to  take  cow's  milk  there  is  a  sudden  increase  in  the 
relative  numbers  of  B.  coli  and  streptococci  and  with  the  addition  of 
starchy  foods  to  the  diet  the  faecal  flora  gradually  comes  to  resemble 
that  of  the  adult. 

In  the  healthy  adult  taking  a  mixed  diet,  the  faecal  flora  consists  for 
the  most  part  of  Gram-negative  bacilli  of  the  type  of  B.  coli.  There  are 
also  many  diplococci,  a  few  small  Gram-positive  bacilli  (B.  bifidus?)  a 
small  number  of  Bact.  Wekhii  and  its  free  spores,  a  few  representatives 
of  the  B.  edematis  group  and  a  variable  number  of  slender  spirochetes. 
Aerobic  plate  cultures  on  agar  or  gelatin  often  bring  to  development 
only  B.  coli.  When  a  vegetarian  diet  rich  in  indigestible  residue  is 
consumed,  the  diplococci  are  much  diminished  in  numbers;  numerous 
large  bacilli,  Bact.  Wekhii  and  B.  subtilis,  take  their  place.  The  con- 
sumption of  excessive  quantities  of  meat  and  starchy  foods  may  lead 
to  a  considerable  increase  in  the  numbers  of  the  Bact.  Wekhii  group  and 
some  of  the  bacteria  of  this  group  may  be  stained  brown  or  blue  with 
iodine  because  of  the  granulose  which  they  contain.  The  bacteria 


MICROORGANISMS   OF   THE   DIGESTIVE   TRACT  599 

normally  present  in  the  faeces  are  produced  almost  altogether  by  multi- 
plication within  the  intestine.  It  is  nevertheless  possible  for  swallowed 
organisms  to  appear  alive  in  the  faeces  even  though  incapable  of  growth 
within  the  digestive  tube. 

The  introduction  of  foreign  organisms  capable  of  multiplication  in 
the  gastro-intestinal  canal  may  lead  to  a  marked  alteration  in  the 
quantitative  relationships  of  the  faecal  bacteria  or  even  to  the  disappear- 
ance of  certain  microbic  forms  previously  present.  Thus  in  cholera, 
the  vibrio  of  this  disease  may  occupy  the  intestinal  canal  so  completely 
that  the  usual  faecal  bacteria  can  no  longer  be  found  with  the  micro- 
scope. By  feeding  acid-resisting  lactose-fermenting  bacteria,  such  as 
Bact.  bulgaricum  along  with  considerable  quantities  of  milk,  it  is  possible 
to  suppress  the  putrefactive  anaerobes,  B.  edematis  group,  which  prefer 
a  neutral  or  alkaline  medium.  The  swallowed  bacteria  are  manifestly, 
therefore,  of  some  importance  in  determining  the  character  of  the  faecal 
flora,  but  they  are,  after  all,  usually  less  important  in  this  respect  than 
the  chemical  composition  of  the  food  itself.  In  every  case  the  original 
intestinal  flora  has  to  be  reckoned  with  as  a  most  essential  element. 

The  daily  excretion*  of  bacteria  in  the  faeces  of  healthy  men,  is, 
on  the  average,  about  33  million  million  bacterial  cells.  The  washed 
and  dried  substance  of  these  bacteria  amounts  to  about  5^  g.  per  day. 
From  one-sixth  to  one-fifth  of  the  weight  of  the  dry  faeces  and  probably 
about  a  third  of  the  moist  faeces  consists  of  bacterial  substance.  The 
nitrogen  carried  away  by  these  faecal  bacteria  represents  a  daily  loss  of 
0.5  to  i.o  g. 

In  addition  to  the  bacteria,  one  often  finds  in  the  faeces  yeasts  and 
protozoa.  Of  the  latter  Entamceba  coli  is  probably  an  almost  constant 
inhabitant  of  the  intestinal  tract  and  its  numbers  are  often  augmented 
in  mild  chronic  digestive  disturbances.  The  flagellates,  Lamblia 
intestinalis  and  Trichomonas  intestinalis  are  found  less  frequently. 
A  few  other  protozoa  occur  in  disease. 

The  physiological  effects  of  the  normal  intestinal  bacteria  are  not 
fully  understood.  Some  observers  have  maintained  that  continued 
life  and  growth  would  be  impossible  without  the  bacteria  of  the  di- 
gestive tract,  ascribing  to  them  an  essential  part  in  the  nutrition  of 
the  body.  The  experiments  of  Cohendyf  seem  now  to  have  disprove n 

*  MacNeal,  Latzer  and  Kerr,  Jour.  Infect.  Diseases,  1909,  VI,  123. 
t  Cohendy,  Annales  de  1'lnstitut  Pasteur,  1912,  XXVI,  io6u 


600  MICROBIOLOGY   OF   FOODS 

this  hypothesis.  There  can  be  no  doubt  that  the  bacteria  do  enter 
intimately  into  intestinal  digestion  and  in  some  instances  bring  about 
changes  beneficial  to  their  host,  such  as  the  digestion  of  cellulose, 
whereas  when  furnished  other  food  they  may  exert  a  harmful  influence, 
as  for  example  in  excessive  intestinal  putrefaction. 

In  diseased  conditions  of  the  gastro-intestinal  tract  one  finds  more 
or  less  well-marked  alterations  in  the  faecal  flora.  These  changes 
include  quantitative  change  in  the  total  bacterial  output,  change  in 
the  proportional  relationships  of  the  various  normal  types  and  finally 
the  appearance  of  new  or  foreign  types  of  organisms,  either  harmless  or 
pathogenic.  In  many  instances  there  is  furthermore  a  distinct  tendency 
for  some  members  of  the  normal  intestinal  flora  to  assume  pathogenic 
properties  and  invade  tissues  rendered  less  resistant  by  disease. 

Among  the  intestinal  microorganisms  which  may  assume  patho- 
genic r61es  at  times  may  be  mentioned  B.  coli,  B.  vulgaris,  Ps.  pyo- 
cyanea,  B.  bifidus,  Bad.  Welchii,  the  streptococci,  micrococci  and 
Trichomonas  intestinalis.  Among  the  definitely  pathogenic  forms  are 
B.  typhosus,  Msp.  comma  (Sp.  cholera  asiaticce),  B.  paratyphosus,  B. 
enteritidis,  Bact.  dysenteries,  Bact.  anthracis,  Bact.  pestis,  Bact.  tuber- 
culosis, Entamceba  dysenteric  (histolytica) ,  Coccidium  hominis  and 
Lamblia  intestinalis. 

The  technical  procedures  necessary  for  the  recognition  of  some 
of  these  organisms  in  the  faeces  and  for  their  isolation  in  pure  culture 
are  in  some  instances  highly  specific.  Thus  if  one  is  searching  for 
B.  bifidus  it  is  best  to  employ  dilution  cultures  in  tall  tubes  of  glucose 
agar  inoculated  with  faeces  of  a  healthy  nursling.  The  same  material 
plated  on  gelatin  will  yield  only  colonies.lcf  B.  coll.  Bact.  Welchii  is  most 
readily  isolated  by  pasteurizing  a  suspension  of  the  faeces  and  introduc- 
ing it  into  blood  broth  or  litmus  milk  in  a  Smith  fermentation  tube. 
The  cholera  organism  is  searched  for  by  introducing  considerable 
quantities  of  faeces  into  flasks  of  pepton-salt  solution  and  transplanting 
from  the  surface  film  after  six  hours  to  new  flasks.  On  account  of 
its  very  rapid  multiplication  in  this  medium  the  cholera  germ,  if 
present,  outstrips  the  other  faecal  bacteria.  Subsequently  it  is  necessary 
to  apply  specific  agglutination  tests  to  the  spirals  thus  obtained  in  order 
to  recognize  them  with  certainty.  The  typhoid  bacillus,  on  the  other 
hand,  is  sought  by  inoculating  media  containing  substances  which 
restrain  bacterial  growth  in  general  without  inhibiting  the  growth  of 


MICROORGANISMS    OF   THE    DIGESTIVE   TRACT  6oi 

B.  typhosus.  Broth  and  agar  containing  brilliant  green,  agar  containing 
fuchsin  and  sulphite  (Endo's  medium)  and  agar  colored  with  eosin  and 
methylene  blue  are  employed  for  this  purpose.  The  tubercle  bacillus 
when  present,  may  sometimes  be  separated  by  digesting  the  faeces  in 
alkali  or  in  antiformin  solution,  washing  the  residue  and  planting  it  on 
Petroff's  medium*  or  injecting  it  into  guinea-pigs.  Entamceba  coll  and 
Enlamczba  dy sentence  should  be  searched  for  with  the  microscope  in 
fresh  warm  faeces  obtained  after  a  dose  of  salts. 

This  brief  mention  of  a  few  procedures  indicates  the  specialized 
character  of  the  microbiological  technic  in  this  field.  The  laboratory 
worker  will  find  it  essential  to  consult  the  general  references  below  and 
to  study  carefully  the  original  papers  bearing  upon  his  field  of  work. 

GENERAL  METHODS  OF  STUDY 

COLLECTION  OF  MATERIAL. — Material  for  microbiological  study  may  be  ob- 
tained from  the  mouth,  fauces  or  pharynx  by  means  of  a  sterile  cotton  swab,  by  the 
ordinary  platinum  loop  or  other  instrument  suitable  for  the  special  purpose  in  view. 
This  material  should  be  examined  promptly,  or,  if  this  is  impossible,  it  should 
at  once  be  spread  upon  slides  for  subsequent  microscopic  study  and,  if  cultures  are 
to  be  made,  it  should  be  suspended  in  sterile  salt  solution,  or  better  in  sterile  ascitic 
fluid,  and  refrigerated  until  the  proper  media  can  be  inoculated.  From  the  stomach, 
fluid  may  be  readily  obtained  through  a  stomach  tube  and  the  contents  of  the 
duodenum  or  of  upper  portions  of  the  small  intestine  may  be  withdrawn  through  the 
slender  duodenal  tube  of  Einhorn.  The  contents  of  the  lower  part  of  the  small 
intestine  and  the  upper  part  of  the  large  intestine  can  be  readily  obtained  only  at 
surgical  operations  upon  the  intestine,  at  autopsies  or  from  individuals  in  whom  an 
intestinal  fistula  has  been  established.  The  contents  of  the  lower  part  of  the  large 
intestine  are  best  collected  by  means  of  a  special  glass  instrument  in  the  case  of 
young  children.  In  older  children  and  adults  a  natural  stool  or  one  obtained 
after  salts  or  other  cathartic  may  be  utilized. 

In  every  instance,  contamination  of  the  material  with  extraneous  organisms  is 
to  be  strictly  avoided  by  careful  sterilization  of  implements  and  receptacles  and 
any  alteration  of  the  specimen  after  collection  must  be  reduced  to  the  minimum 
by  examining  it  promptly,  although,  for  some  purposes  the  use  of  refrigerated  speci- 
mens may  be  permitted. 

The  quantity!  of  microbic  cells  present  may  be  ascertained  by  numerical  count 
of  those  present  in  an  accurately  measured  portion  of  the  material,  or  if  they  are 
very  abundant  they  may  be  physically  separated  out  from  a  weighed  portion 
by  fractional  sedimentation  in  the  centrifuge,  after  which  they  are  dried  and  weighed 
(method  of  Strasburger). 

*Petroff.  Journ.  Exp.  Med.,  1915,  XXI,  38. 

t  For  detailed  directions  concerning  quantitative  methods  as  applied  to  the  study  of  faeca 
bacteria,  see  MacNeal,  Latzer  and  Kerr.  Journ.  Infect.  Diseases,  1909,  VI,  123;  ibid.,  1909, 
VI.  S7I. 


602 


MICROBIOLOGY   OF  FOODS 


A  preliminary  classification  of  the  recognizably  different  kinds  of  microbes 
should  be  made  by  microscopic  examination  of  film  preparations  stained  (i)  with 
LoefHer's  methylene  blue,  (2)  by  Gram's  method,  (3)  by  the  Ziehl-Neelsen  method 
and  (4)  simply  withLugol's  solution.  It  is  best  to  count  from  500  to  1,000  microbic 
cells  as  they  are  met  with  in  successive  microscopic  fields  and  to  classify  them 
according  to  form,  size  and  structural  details  brought 
out  by  the  different  stains.  Permanent  records  and,  if 
possible,  permanent  mounted  preparations  should  be 
preserved,  so  that  the  microbes  subsequently  brought 
to  development  in  the  cultures  may  be  identified  with 
some  of  those  present  in  the  microscopic  picture  of 
the  original  material. 

Cultures  are  best  made  upon  a  quantitative  basis, 
employing  for  inoculation  measured  amounts  of  accur- 
ately prepared  dilutions  of  the  original  material.  There 
is  no  single  culture  medium  or  method  which  can  be 
relied  upon  to  give  any  approximate  conception  of  the 
numerical  relations  of  the  microbes  of  the  digestive  tract. 
Each  cultural  method  necessarily  favors  certain  species 
present  in  the  mixture  and  allows  others  to  develop 
only  poorly  or  not  at  all.  Adequate  information  con- 
cerning the  quantitative  relationships  is  obtained  only 
by  comparing  the  results  of  the  culture  work  with  the 
direct  quantitative  estimations  and  by  fitting  the  cul- 
tural results  into  the  original  microscopic  picture.  A 
great  variety  of  culture  media  and  culture  methods, 
aerobic,  anaerobic  and  micro-aerophilic,  must  be  em- 
ployed in  making  even  an  incomplete  general  survey  of 
the  microbes  from  any  portion  of  the  digestive  tract. 
For  the  detection  of  certain  single  species,  on  the  other 
hand,  one  may  sometimes  rely  upon  a  single  medium, 
such  as  blood-agar  for  the  streptococci  of  the  mouth, 
LoefHer's  serum  for  diphtheria  bacilli  in  the  pharynx  01  blood-broth  in  fermenta- 
tion tube  for  spores  of  B.  welchii  in  the  faeces.  Thus  the  numerous  time-consuming 
procedures  may  be  very  much  abridged  and  many  of  them  may  well  be  omitted 
when  one  wishes  to  ascertain  merely  the  presence  or  absence  of  a  certain  single 
species  of  microbe. 

GENERAL  REFERENCES  ' 

Schmidt  und  Strasburger,  Die  Faeces  des  Menschen,  IV  "  Auflage,  Berlin,  1914. 

Kuester,  Die  Flora  der  normalen  Mundhohle,  Kolle  und  Wassermann,  Hand- 
buch,  IP  Auflage,  Jena,  1913,  VI,  435-449. 

Kuester,  Die  Bedeutung  der  normalen  Darmbakterien  fur  den  gesunden  Men- 
schen, Kolle  und  Wassermann,  Handbuch,  II"  Auflage,  Jena,  1913,  VI,  468-482. 


FIG.  155. — Two  types 
of  instrument  for  obtain- 
ing faeces  from  infants  for 
bacteriological  examina- 
tion. (After  Schmidt  and 
Strasburger.) 


DIVISION  VI 

MICROBIOLOGY  OF  ALCOHOLIC  FERMENTATION  AND 
DERIVED  PRODUCTS* 


CHAPTER  I 

WINE 

Wine  may  be  defined  shortly  as  the  product  of  the  alcoholic  fer- 
mentation of  sound,  ripe  grapes  and  the  usual  cellar  treatment. 

The  classifications  of  wines  are  numerous  and  the  varieties  in- 
numerable. They  may  be  separated,  however,  into  a  few  main  groups, 
depending  on  chemical  composition  and  methods  of  manufacture. 
Dry  wines  are  those  in  which  practically  all  the  sugar  has  been  re- 
moved by  fermentation;  sweet  wines,  those  in  which  enough  sugar 
remains  or  is  added  to  be  noticeable  to  the  taste ;  fortified  wines,  those 
that  have  received  an  addition  of  distilled  wine  spirits;  and  sparkling 
wines,  those  highly  charged  with  carbon  dioxide,  produced  by  supple- 
mentary fermentation  in  the  bottle.  Each  of  these  groups  includes 
white  wines  made  from  the  expressed  juice  of  the  grape,  and  red  wines 
made  from  both  the  juice  and  skins  of  red  grapes. 

Wine  in  the  proper  sense  is  therefore  produced  exclusively  from 
fresh  grapes.  Much  so-called  wine  is  made  in  many  countries  from 
dried  grapes  or  mixtures  of  grapes  and  other  fruits  with  sugary  materials 
of  various  kinds  and  various  coloring  and  flavoring  substances.  Some 
contain  no  grapes  at  all.  In  most  countries,  these  beverages  cannot 
be  sold  without  some  qualifying  designation,  such  as  modified,  amelio- 
rated, or  imitation  wine,  or  piquette,  plum  wine,  gooseberry  wine,  etc. 

GRAPE  JUICE  AND  WINE  AS  CULTURE  MEDIA 

Grape  juice,  known  technically  as  must,  is  a  sugary,  acid,  organic  so- 
lution very  favorable  to  the  growth  of  yeasts  and  of  many  other  fungi, 
but  unfavorable  to  most  bacteria.  Wine  is  of  a  similar  composition  but 
contains  alcohol  instead  of  sugar  and  is,  therefore,  less  favorable  to  the 

•  Prepared  by  F.  T.  Bioletti. 

603 


604 


MICROBIOLOGY   OF   ALCOHOLIC  FERMENTATION 


growth  of  most  microorganisms.  Both  liquids  are  of  highly  complex 
composition.  Their  character  as  culture  media  is  indicated  by  the 
following  table: 

COMPOSITION  OF  MUST  AND  DRY  \VINE 


Must 

Wine 

Specific  gravity  

i  .  0600  to  i  .  1090 

o  9850  to  i  .  oooo 

Fermentable  sugar 

12  o        to  25  o  per  cent 

o  to  o  5  per  cent 

Alcohol  by  volume  

none 

8.0  to  15.0  per  cent 

Acidity  (as  tartaric) 

05        to  i  25  per  cent 

°  35  to  i  o  per  cent 

Nitrogenous  matters 
(soluble) 

02        to    o  4  per  cent 

variable  but  small 

Tannin  

Traces 

traces  to  0.30  per  cent 

14    to  4  o    per  cent 

Ash.. 

0.20        to  0.70  per  cent 

o.n  to  o.so  oer  cent 

Fortified  wines  (sweet  wines  are  usually  fortified)  usually  con- 
tain enough  alcohol  to  make  them  practically  antiseptic  to  all 
microorganisms. 

THE  MICROORGANISMS  FOUND  ON  GRAPES 

On  the  surfaces  of  grapes,  as  they  are  brought  to  the  cellar,  may  be 
found  any  of  the  bacteria  and  fungi  usually  carried  by  the  air  and  by 
insects.  Many  of  these  are  incapable  of  growing  in  grape  must,  and 
are,  therefore,  without  effect  on  the  wine. 

MOLDS. — The  spores  of  the  common  saprophytic  molds,  Penicil- 
lium,  Dematium,  Aspergillus,  Mucor,  are  always  present  in  abundance, 
and  they  find  in  must  excellent  conditions  for  development.  Botrytis 
cinerea,  a  facultative  parasite  of  the  leaves  and  fruit  of  the  vine,  is  also 
nearly  always  present  in  larger  or  smaller  quantities.  All  these  molds 
are  harmful  to  the  grapes  and  the  wine.  Some  of  them,  such  as  Penicil- 
lium,  may  give  a  disagreeable,  moldy  taste  to  the  wine,  sufficient  to 
spoil  its  commercial  value.  Others,  such  as  some  Mucor s  and  Asper- 
gilli,  may  injure  the  wine  but  slightly  except  by  destroying  sugar  and 
diminishing  the  alcohol.  Dematium  pullulans  may  produce  a  slimy 
condition  in  weak  white  musts  and  inost  of  them  may  injure  the 
brightness  and  flavor  to  some  extent. 

On  sound  ripe  grapes  these  molds  occur  in  comparatively  small 
numbers  and  being  in  the  spore  or  dormant  condition  they  are  unable 


WINE  605 

to  develop  sufficiently  to  injure  the  wine  under  the  conditions  of  proper 
wine  making.  On  grapes  which  are  injured  by  diseases,  rain  or  insects, 
tliey  may  be  present  in  sufficient  quantities  to  spoil  the  grapes  before 
they  are  gathered.  On  sound  grapes  which  are  gathered  and  handled 
carelessly,  they  may  develop  sufficiently  before  fermentation  to  injure 
or  spoil  the  wine. 

An  exception  to  the  generally  harmful  effect  of  these  molds  is 
Botrytis  cinerea  (Sclerotinia  fuckeliana)  which  under  certain  circum- 
stances may  have  a  beneficial  action.  When  the  conditions  of  tem- 
perature and  moisture  are  favorable,  this  mold  will  attack  the  skin  of 
the  grape,  facilitating  evaporation  of  water  from  the  pulp.  This 
results  in  a  concentration  of  the  juice.  The  mycelium  then  penetrates 
the  pulp,  consuming  both  sugar  and  acid,  principally  the  latter.  The 
net  result  is  an  increase  in  the  percentage  of  sugar  and  a  decrease  in 
that  of  acid.  This,  where  grapes  ripen  with  difficulty,  is  an  advantage, 
as  no  moldy  flavor  is  produced.  Two  harmful  effects,  however,  follow : 
the  growth  of  the  mold  results  in  the  destruction  of  a  certain  amount  of 
material,  and  a  consequent  loss  of  quantity,  which  is,  in  certain  circum- 
stances, more  than  counterbalanced  by  an  increase  in  quality  (wines 
of  the  Rhine,  Sauternes) ;  again,  an  oxidase  is  produced  which  tends  to 
destroy  the  color,  brightness  and  flavor  of  the  wine.  This  can  be 
counteracted  by  the  judicious  use  of  sulphurous  acid. 

YEASTS. — The  true  yeasts  occur  much  less  abundantly  on  grapes 
than  the  molds.  Until  the  grapes  are  ripe  they  are  practically  absent, 
as  first  shown  by  Pasteur.  Later,  they  gradually  increase  in  number 
and  on  very  ripe  grapes  often  become  abundant.  In  all  cases  and  at  all 
seasons,  however,  their  numbers  are  much  inferior  to  those  of  the  molds 
and  pseudo-yeasts.  The  cause  of  this  seems  to  be  that  in  the  vineyard 
the  common  molds  find  conditions  favorable  to  their  development  at 
nearly  all  seasons  of  the  year,  but  yeasts  only  during  the  vintage  season. 

Investigations  of  Hansen,  Wortmann  and  others  show  that  yeasts 
exist  in  the  soil  of  the  vineyard  at  all  times,  but  in  very  varying  amounts. 
For  a  month  or  two  following  the  vintage,  a  particle  of  soil  added  to  a 
nutritive  solution  contains  so  much  yeast  that  it  acts  like  a  leaven.  For 
the  next  few  months,  the  amount  of  yeast  present  decreases  until  a 
little  before  the  vintage,  when  the  soil  must  be  carefully  examined  to 
find  any  yeast  at  all.  As  soon  as  the  grapes  are  ripe,  however,  any 
rupture  of  the  skin  of  the  fruit  will  offer  a  favorable  nidus  for  the 


606  MICROBIOLOGY   OF  ALCOHOLIC   FERMENTATION 

development  and  increase  of  any  yeast  cells  which  reach  it.  Where 
these  first  cells  come  from  has  not  been  determined,  but  as  there  are 
still  a  few  yeast  cells  in  the  soil,  they  may  be  brought  by  the  wind,  or 
bees  and  wasps  may  carry  them  from  other  fruits  or  from  their  hives 
and  nests. 

The  increase  of  the  amount  of  yeast  present  on  the  ripe  grapes  is 
often  very  rapid  and  seems  to  have  (according  to  Wortmann)  a  direct 
relation  to  the  abundance  of  wasps.  These  insects,  passing  from  vine 
to  vine,  crawling  over  the  bunches  to  feed  on  the  juice  of  ruptured 
berries,  soon  inoculate  all  exposed  juice  and  pulp.  New  yeast  cultures 
are  thus  produced,  and  the  resulting  yeast  cells  quickly  disseminated 
over  the  skins  and  other  surfaces  visited. 

The  more  unsound  or  broken  grapes  present,  and  the  more  honey- 
dew  or  dust  adhering  to  the  skin,  the  larger  the  amount  of  yeast  will 
be.  The  same  is  true,  however,  also  of  molds  and  other  organisms. 

In  the  older  wine-making  districts,  much  of  the  yeast  present  on  the 
grapes  will  consist  of  the  true  wine  yeast,  S.  ellipsoideus.  The  race  or 
variety  of  this  yeast  will  differ,  however,  in  different  districts.  Usually 
several  varieties  will  be  found  in  each  district.  The  idea  prevalent  at 
one  time,  that  each  variety  of  grapes  has  its  own  variety  of  yeast  seems 
to  have  been  disproved,  though  there  seems  to  be  some  basis  for  the 
idea  that  grapes  differing  very  much  in  composition,  varying  in  acidity 
and  tannin  contents,  may  vary  also  in  the  kind  of  yeast  present. 
Several  varieties  of  S.  ellipsoideus  may  occur  on  the  same  grapes.  In 
new  grape-growing  districts,  where  wine  has  never  been  made,  5. 
ellipsoideus  may  be  completely  absent. 

Besides  the  true  wine  yeast,  other  yeasts  usually  occur.  The  com- 
monest forms  are  cylindrical  cells  grouped  as  5.  pasteurianus.  These 
forms  are  particularly  abundant  in  the  newer  districts  where  they  may 
take  a  notable  part  in  the  fermentation.  Their  presence  in  large 
numbers  is  always  undesirable  and  results  in  inferior  wine.  Many 
other  yeasts  may  occur  occasionally  and  are  all  more  or  less  harmful. 
Some  have  been  noted  as  producing  sliminess  in  the  wine.  Many  of 
these  yeasts  produce  little  or  no  alcohol  and  will  grow  only  in  the 
presence  of  oxygen. 

Pseudo-yeasts. — Yeast-like  organisms  producing  no  endospores 
always  occur  on  grapes.  Their  annual  life-cycle  and  distribution  are 
similar  to  those  of  the  true  yeasts,  but  some  of  them  are  much  more 


WINE  607 

abundant  than  the  latter.  They  live  at  the  expense  of  the  food 
materials  of  the  must  and  when  allowed  to  develop  cause  cloudiness 
and  various  defects  in  the  wine. 

The  most  important  and  abundant  is  the  apiculate  yeast,  S.  api- 
culatus.  According  to  Lindner  this  is  a  true  yeast,  producing  endo- 
spores.  The  cells  of  this  organism  are  much  smaller  than  those  of 
S.  ellipsoideus  and  very  distinct  in  form.  In  pure  culture  these  cells 
show  various  forms,  ranging  from  ellipsoidal  to  pear-shaped  (apiculate 
at  one  end)  and  lemon-shaped  (apiculate  at  both  ends).  These  forms 
represent  simply  stages  of  development.  The  apiculations  are  the 
first  stage  in  the  formation  of  daughter  cells,  the  ellipsoidal  cells,  the 
newly  separated  daughter  cells,  which  later  produce  apiculations  and 
new  cells  in  turn. 

Many  varieties  of  this  yeast  occur,  as  in  the  case  of  S.  ellipsoideus. 
They  are  widely  distributed  in  nature,  occurring  on  most  fruits,  and 
are  particularly  abundant  on  acid  fruits  such  as  grapes.  Apiculate 
yeast  appears  on  the  partially  ripe  grapes  before  the  true  wine  yeast 
and  even  on  ripe  grapes  is  more  abundant  than  the  latter.  The  rate 
of  multiplication  of  this  yeast  is  very  rapid  under  favoring  conditions 
and  much  exceeds  that  of  wine  yeast.  The  first  part  of  the  fermenta- 
tion, especially  at  the  beginning  of  the  vintage  and  with  acid  grapes, 
is,  therefore,  often  almost  entirely  the  work  of  the  apiculate  yeast. 

The  amount  of  alcohol  produced  by  this  yeast  is  about  4  per  cent, 
varying  with  the  variety  from  2  to  6  per  cent.  When  the  fermentation 
has  produced  this  amount  of  alcohol  the  activity  of  the  yeast  slackens 
and  finally  stops,  allowing  the  more  resistant  ellipsoideus  to  multiply 
and  finish  the  destruction  of  the  sugar.  The  growth  of  S.  apiculatus, 
however,  has  a  deterring  effect  on  that  of  the  true  wine  yeast  so  that 
where  much  of  the  former  has  been  present  during  the  first  stages  of 
fermentation  the  latter  often  fails  to  eliminate  all  the  sugar  during  the 
last  stages. 

When  the  apiculate  yeast  has  had  a  large  part  in  the  fermentation, 
the  wines  are  apt  to  retain  some  unfermented  sugar  and  are  open 
to  the  attacks  of  disease-producing  organisms.  Their  taste  and  color 
are  defective,  often  suggestive  of  cider,  and  they  are  difficult  to  clarify. 
This  yeast  attacks  the  fixed  acids  of  the  must,  the  amount  of  which  is, 
therefore,  diminished  in  the  wine,  while  on  the  other  hand  the  volatile 
acids  are  increased. 


608  MICROBIOLOGY   OF  ALCOHOLIC   FERMENTATION 

Many  other  yeast-like  organisms  may  occur  on  grapes,  but,  under 
ordinary  conditions,  fail  to  develop  sufficiently  in  competition  with 
apiculatus  to  have  any  appreciable  effect  on  the  wine.  Most  of  them 
are  small  round  cells,  classed  usually  as  Torulce.  They  destroy  the 
sugar  but  produce  little  or  no  alcohol. 

A  group  of  similar  forms,  known  collectively  as  Mycoderma  vini, 
occurs  constantly  on  the  grapes.  These,  being  strongly  aerobic,  do  not 
develop  in  the  fermenting  vat,  but  under  favoring  conditions  may  be 
harmful  to  the  fermented  wine. 

BACTERIA  of  many  kinds  occur  on  grapes  as  on  all  surfaces  exposed 
to  the  air.  Most  of  these  are  unable  to  develop  in  solutions  so  acid  as 
grape  juice  or  wine.  Of  the  acid-resisting  kinds,  a  number  may  cause 
serious  defects  and  even  completely  destroy  the  wine.  These,  the 
"disease-producing  bacteria"  of  wine,  are  mostly  anaerobic  and  can 
develop  only  after  the  grapes  are  crushed  and  the  oxygen  of  the  must 
exhausted  by  other  organisms.  Practically  all  grape  must  contains 
some  of  these  bacteria,  which,  unless  the  work  of  the  wine  maker  is 
properly  done,  will  seriously  interfere  with  the  work  of  the  yeast,  thus 
causing  injury  to  the  wine.  The  only  bacteria  which  may  injure  the 
grapes  before  crushing  are  the  aerobic,  acetic  bacteria,  which  may 
develop  on  injured  or  carelessly  handled  grapes  sufficiently  to  interfere 
with  fermentation  and  seriously  impair  the  quality  of  the  wine. 

THE  MICROORGANISMS  FOUND  IN  WINE 

Wine  microorganisms  may  be  conveniently  divided  into  two  groups: 
those  which  grow  only  in  the  presence  of  notable  supplies  of  free 
oxygen,  and  those  which  require,  or  grow  better  in,  the  absence  of  free 
oxygen. 

AEROBIC  ORGANISMS.  Mycodermce. — If  a  normal  wine,  especially 
one  strong  in  alcohol,  is  left  with  its  surface  exposed  to  the  air,  it  will 
usually,  in  a  few  days,  be  covered  with  a  whitish  film,  thin  and  smooth 
at  first  but  gradually  becoming  thicker  and  finally  rough  and  plicate. 
This  is  what  is  known  to  wine-makers  as  "  wine  flowers"  This  film  con- 
sists of  yeast-like  cells,  somewhat  longer  and  more  cylindrical  than  S. 
ellipsoideus,  reproducing  by  budding  and  forming  large  aggregations. 

Pure  cultures  show  that  there  are  many  varieties  of  this  organism 
differing  in  the  color  and  texture  of  the  film,  in  the  cloudiness  of  the 
liquid  and  in  the  character  of  the  deposit.  They  are  called  collectively 


WINE  609 

Mycoderma  vini,  though  one  form  which  has  been  found  to  produce 
endospores  has  been  called  S.  anomalus. 

These  organisms  are  strongly  aerobic  and  can  develop  only  on  the 
surface  in  full  contact  with  the  air.  They  are  a  serious  enemy  to  the 
wine,  rendering  it  insipid  and  cloudy.  They  attack  the  extract,  fixed 
acids,  and  alcohol,  producing  at  first  volatile  acids  and  finally  causing 
complete  combustion  of  the  organic  matters  to  carbon  dioxide  and  water, 
destroying  the  wine  completely. 

Acetic  Bacteria. — The  film  formed  on  wines  exposed  to  the  air, 
especially  on  those  of  low  alcoholic  content,  will  often  differ  from  that 
due  to  Mycoderma  vini.  It  will  be  thinner,  smoother  and  consist  of 
bacteria.  These  are  the  vinegar  bacteria  described  on  page  637. 
They  grow  not  only  on  the  wine  at  the  expense  of  the  alcohol,  but  on 
crushed  grapes  and  must  at  the  expense  of  the  sugar,  producing  acetic 
acid  in  both  cases. 

Acetic  acid  in  small  amounts  is  produced  by  the  yeast  and  is  a 
normal  constituent  of  wine.  Unless  in  excess  its  effect  is  not  injurious. 
There  may  be  present  from  0.12  g.  in  100  c.c.  in  light  white  wine  to  0.14 
g.  in  a  heavy  red  wine  without  deterioration  of  quality.  In  sweet  wines, 
even  a  somewhat  larger  amount  may  be  present  without  causing  injury. 

Much  larger  amounts  are  injurious  in  two  ways.  When  the  acetic 
acid  is  perceptible  to  the  taste,  the  wine  is  spoiled.  When  an  abnormal 
amount  of  acetic  acid  is  produced  before  or  during  fermentation  it 
stops  or  interferes  with  the  work  of  the  yeast.  In  such  cases,  the  wine 
"  sticks,"  that  is,  fails  to  eliminate  all  its  sugar  and  becomes  especially 
open  to  the  attacks  of  other  bacteria. 

Wines  high  in  alcohol  are  less  liable  to  acetic  fermentation  than 
weaker  wines.  Sound  wines  containing  over  14  per  cent  by  volume 
of  alcohol  are  almost  immune,  but  such  wines  may  be  spoiled  during 
fermentation  by  the  growth  of  acetic  bacteria  on  the  exposed  float- 
ing "cap"  of  pomace  or  on  the  crushed  grapes,  especially  at  high 
temperatures. 

ANAEROBIC  ORGANISMS  (Facultative  and  Obligate). — Some  of  the 
worst,  most  frequent,  and  most  difficult  diseases  and  defects  of  wine 
to  treat  are  due  to  organisms  which  develop  only  in  the  absence 
of  oxygen.  These  organisms  are  all  bacteria  and  appear  to  include  a 
large  number  of  forms,  though,  owing  to  difficulties  of  isolation  and 
culture,  the  different  forms  have  not  been  well  studied  or  described. 

39 


6io 


MICROBIOLOGY   OF  ALCOHOLIC   FERMENTATION 


Slime-forming  Bacteria. — Musts  and  wines  become  slimy  rarely 
through  the  action  of  Dematium  pullulans  (Wortmann)  and  wild  yeast 
(Meisner)  in  the  presence  of  oxygen  but  more  frequently  through  the 
action  of  bacteria.  In  most  cases  only  young  wines  after  fermentation 
and  when  contained  in  closed  casks  or  bottles  exhibit  this  defect.  A 
slimy  wine  has  an  oily  appearance,  pours  without  splashing  and  in 
extreme  cases,  becomes  cloudy  and  will  hang  from  a  glass  rod  in  strings. 

In  such  wines,  the  microscope  reveals  large  numbers  of  almost 
spherical  or  more  or  less  elongated  bacteria  in  long  chains.  Some  ob- 
servers have  noticed  a  diplococcus  and  a  sarcina.  Kayser  and  Manceau 


/.x.i/'^r:;^-'^ 
^> "'-  4/N  v»*; 


FIG.  1 56. — Bacteria  of  slimy  wine.     A,B,Ct  Pure  cultures  of  various  forms;  D,  muci- 
•laginous  sheath  of  slime  bacteria.     (After  Kayser  and  Manceau.}    J 

have  recently  investigated  the  subject  very  thoroughly  and  described  a 
number  of  forms  which  are  mostly  short  rods  of  from  1/4  to  2/4  by  0.7/4 
to  1.2/4.  One  large  form,  3/4  to  4/4  X  1.6/4  to  1.7/4  was  also  noted. 
They  all  form  chains,  usually  of  considerable  length.  They  all  produce 
an  abundant  slimy  sheath  and  stain  easily  with  carbol-fuchsin  and  other 
aniline  dyes  and  are  Gram-positive  (Fig.  156). 

These  bacteria  attack  the  sugar  but  neither  the  glycerin  nor  the 
alcohol  and  produce  mannit,  carbon  dioxide,  lactic  and  acetic  acids  and 
ethyl  alcohol.  The  disease  is  usually  not  serious  and  disappears  under 
the  ordinary  cellar  treatment.  Alcohol  above  13  per  cent,  free  tartaric 
acid,  tannin  and  sulphurous  acid  in  small  amounts  prevent  their 
growth. 

Propionic  and  Lactic  Bacteria. — The  most  serious  and  perhaps 
the  commonest  disease  of  wines  is  characterized  by  persistent  cloudi- 
ness, disagreeable  odors  and  flavors,  increase  of  volatile  acid  and 
injury  to  the  color  or  its  complete  destruction.  Wines  affected  are 


WINE 


611 


characterized  commonly  as  mousey,  lactic  or  turned  wines  (Pousse  and 
Tourne  of  the  French). 

The  disease  is  due  to  bacteria.  Enormous  numbers  are  readily 
revealed  by  the  microscope  in  badly  affected  wines.  There  seem  to 
be  several  or  many  closely  related  forms,  all  short  rod-shaped,  isolated 
in  the  first  stages  of  the  disease,  but  later  forming  chains  or  filaments 
of  various  lengths.  The  most  noticeable  change  caused  in  the  com- 
position of  the  wine  is  the  decrease  of  fixed  and  increase  of  volatile 
acidity.  The  tartaric  acid  and  tartrates  are  destroyed,  and  carbonic, 
acetic,  lactic,  propionic  and  other  acids  formed. 


\ 

L 


FIG.  157. — Bacteria  of  wine  diseases.  A,  Bacteria  of  "  turned  wine,"  young  wine 
(After  Bioletti);  B,  bacteria  of  "turned  wine,"  old  wine  (Afler  Biolelti);  C,  mannitic 
bacteria  (After  Maze  and  Pacottef);  D,  bacteria  of  "bitter  wine"  (After  Pacoltet). 

Light  wines  of  low  acidity  are  most  subject  to  this  disease  which 
may  be  prevented  by  measures  which  increase  the  acidity  and  alcohol, 
by  rapid  and  complete  defecation  and  attenuation  of  the  wine  with 
the  proper  use  of  sulphurous  acid,  and  finally  by  timely  filtration  and 
pasteurization.  Wines  noticeably  affected  can  be  used  only  for  dis- 
tilling; those  badly  affected  are  valueless. 

Mannitic  Bacteria. — Very  sweet  grapes  of  low  acidity  in  hot  climates 
are  subject  during  fermentation  to  a  similar  trouble  characterized  by 
increase  of  volatile  acids  and  a  persistent  cloudiness  and  a  vapid 
sweet-sour  taste.  The  disease  is  commonly  confused  with  the  preceding 
but  is  caused  by  bacteria  of  different  forms.  The  form  described  by 
Gayon  is  a  very  fine  short  rod  which  does  not  unite  in  filaments.  It 
attacks  the  sugar,  especially  the  levulose,  producing  volatile  acids  and 


6l2  MICROBIOLOGY  OF  ALCOHOLIC   FERMENTATION 

mannit.  The  latter  may  reach  over  2  per  cent  and  the  former  5  per 
cent,  giving  a  sweet-sour  wine  which  is  completely  spoiled. 

The  bacteria  grow  abundantly  only  at  high  temperatures  approach- 
ing 40°  and  can  be  controlled  by  cool  fermentation,  increase  of  acidity 
and  proper  use  of  sulphurous  acid. 

Butyric  Bacteria. — In  the  cooler  climates,  wines,  especially  old  red 
wines  in  bottles,  often  become  bitter.  This  trouble  is  due  to  com- 
paratively large  rod-shaped  bacteria,  first  described  by  Pasteur. 
The  cells  remain  united  in  angular  filaments,  short  at  first,  but  be- 
coming longer  and  finally  thicker  with  age  by  incrustations  of  coloring 
matter. 

The  tannin,  coloring  matter,  and  glycerin  of  the  wine  are  attacked, 
acetic  and  butyric  acids  being  formed.  In  small  amounts  the  bacteria 
do  little  or  no  harm,  in  larger  amounts  they  may  spoil  the  wine.  Means 
which  increase  the  alcohol,  tannin  and  acidity  diminish  the  liability  to 
the  disease.  Prompt  attenuation  and  clarification  and  in  extreme  cases 
pasteurization  will  cure  wines  not  too  badly  affected. 

All  the  above  anaerobic  bacteria  of  wine  diseases  probably  exist  in 
most  wines.  Which  develop  most,  or  whether  any  develop  sufficiently 
to  injure  the  wine  depends  on  conditions,  chiefly  the  composition  of  the 
must  and  the  temperature  at  which  the  wine  is  fermented  or  stored. 
Most  diseased  wines  show  a  mixed  infection  of  several  forms.  Re- 
cently W.  V.  Cruess  has  found  bacteria  in  wine  containing  twenty 
per  cent  of  alcohol.  These  bacteria  were  living  and  causing  cloudi- 
ness and  increasing  the  volatile  acids. 

CONTROL  OF  THE  MICROORGANISMS 

Given  grapes  of  suitable  composition,  the  quality  of  the  wine 
depends  on  the  work  of  microorganisms.  The  art  of  the  wine-maker 
consists  almost  entirely  in  the  control  of  these  microorganisms.  His 
success  in  facilitating  the  work  of  the  useful  form  (true  wine  yeast) 
and  in  preventing  or  hindering  the  work  of  injurious  forms  determines 
the  quality  of  his  product. 

BEFORE  FERMENTATION.— On  the  skins  of  sound  ripe  grapes  as  they 
hang  in  the  vineyard,  the  microorganisms  are  comparatively  few  and  in 
an  inactive  condition.  When  proper  methods  are  used  they  cannot 
injure  the  wine.  On  broken  or  injured  grapes,  the  number  is  greater 


WINE  613 

and  the  forms  more  active.     If  many  such  grapes  occur  they  should 
not  be  mixed  with  the  sound  grapes  if  the  best  wine  is  to  be  made. 

Care  should  be  taken  to  avoid  unnecessary  bruising  of  the  fruit  if  it  cannot  be 
worked  immediately.  Molds,  wild  yeasts  and  acetic  bacteria  multiply  rapidly  on 
grapes  wet  with  juice. 

The  sooner  the  grapes  can  be  crushed  and  placed  in  the  fermenting  vat  or  pressed 
the  easier  it  is  to  obtain  a  sound  fermentation. 

Cleanliness  is  essential.  Grapes,  which  are  gathered  in  moldy,  vinegar-sour 
boxes,  hauled  in  dirty  wagons  or  cars,  and  passed  through  dirty  crushers,  conveyors 
and  presses,  may  be  so  completely  infected  with  injurious  germs  that  it  is  impossible 
to  obtain  a  good  fermentation.  The  most  injurious  forms  of  dirt  are  must,  grapes, 
or  wine,  which  have  been  allowed  to  become  moldy  or  vinegar-sour. 

Dust  or  soil  is  less  injurious  and,  if  excessive,  may  often  be  removed  by  sprink- 
ling, especially  is  this  true  if  the  grapes  are  too  sweet  and  require  dilution.*  Washing 
with  antiseptics  is  not  permissible.  A  weak  solution  of  potassium  metabisulphite 
might  be  used  with  benefit  if  it  were  not  for  the  difficulty  of  regulating  the  amount  of 
sulphurous  acid  entering  the  fermenting  vessel. 

If  the  grapes  have  to  be  kept  for  some  time  before  crushing,  they  should  be  kept 
as  cool  as  possible  to  delay  the  growth  of  molds.  Gathering  in  the  cool  of  the  morn- 
ing is  desirable  and  if  grapes  are  gathered  when  warm  they  should  be  left  in  boxes 
to  cool  off  during  the  night  whenever  possible.  If  the  grapes  are  cool  when  they 
reach  the  fermenting  vat,  they  will  neutralize  a  certain  proportion  of  the  heat 
of  fermentation,  and  the  difficulty  of  avoiding  injuriously  high  temperatures  is 
diminished. 

However  carefully  the  grapes  are  handled,  a  certain  amount  of  dust  containing 
germs  and  other  injurious  matters  will  reach  the  vats  and  presses.  In  the  manu- 
facture of  white  wines,  especially,  it  is  desirable  to  get  rid  of  these  matters  before 
fermentation.  This  is  best  accomplished  by  settling  and  decantation. 

As  the  juice  runs  from  the  press,  it  is  pumped  into  a  settling  tank  or  cask.  If  it 
is  cold,  below  15°,  and  of  full  normal  acidity,  the  impurities  may  settle  in  twenty  to 
forty-eight  hours.  If  the  temperature  is  higher  than  15°  and  the  acidity  low, 
molds  and  yeasts  will  develop  or  fermentation  will  start  and  prevent  settling.  A 
slight  sulphuring  with  the  fumes  of  burning  sulphur  or  with  a  solution  of  potassium 
metabisulphite  is  therefore  usually  necessary.  The  sulphuring  should  be  as  light 
as  possible  with  acid  musts  as  it  tends  to  preserve  the  fixed  acids.  For  the  same 
reason  it  benefits  musts  of  low  acidity.  In  from  twelve  to  twenty-four  hours,  the 
must  is  purged  of  all  its  gross  impurities  including  dust,  and  solid  particles  de- 
rived from  the  skins  and  the  stems  and  pulp  of  the  grapes.  It  may  be  slightly 
cloudy  or  nearly  clear.  It  can  then  be  drawn  off  into  clean  casks  and  fermen- 
tation started  with  yeast.  The  microorganisms  settle  only  in  part  but  they  are 
all  paralyzed  temporarily. 

This  defecation  is  of  great  value,  ridding  the  must  of  substances  that  would 
affect  the  flavor  of  the  wine  in  the  heat  of  fermentation  and  eliminating  the  excess 

*  Formerly  a  decision  of  the  U.  S.  Dept.  of  Agriculture  forbade  the  use  of  the  term  "pure 
'wine"  when  water  in  even  the  smallest  quantities  had  been  used.  By  the  federal  law  of  1916 
dilution  with  water  up  to  35  per  cent  is  allowed. 


6 14  MICROBIOLOGY   OF   ALCOHOLIC   FERMENTATION 

of  protein  matters  that  would  serve  as  food  for  injurious  bacteria.  Centrifugal 
machines  have  been  devised  to  hasten  the  process  of  defecation,  but  their  work  is 
imperfect. 

Sterilization  by  heat  has  been  tried  for  the  same  purpose  but  with  indifferent 
success.  High  heating  caramelizes  part  of  the  sugar  and  oxidizes  the  must,  thus 
injuring  the  flavor.  Discontinuous  heating  at  lower  temperatures  in  an  atmosphere 
of  carbon  dioxide  is  preferable  but  troublesome  and  expensive.  All  methods  have  the 
defect  of  extracting  undesirable  substances  from  the  solid  matters  which  are  heated 
with  the  must. 

Chemical  sterilization  is  still  less  practicable.  No  substance  could  be  used  for 
this  purpose  except  sulphur  dioxide;  this  used  in  sufficient  quantities  would  seriously 
injure  the  flavor  of  the  wine.  The  effect  would  be  totally  different  from  that  of  the 
small  quantities  used  in  defecation. 

All  the  methods  discussed  have  for  their  object  the  diminution  or  elimination  of 
microorganisms  of  all  kinds.  With  the  injurious  forms  the  true  yeast  is  also  re- 
moved. The  more  perfect  these  methods,  the  more  necessary  it  is  to  add  wine 
yeast.  Without  this  addition,  in  fact,  all  these  precautions  may  result  in  harm,  for 
the  wine  yeast,  being  present  in  much  smaller  numbers  than  many  of  the  injurious 
forms,  may  be  completely  removed  while  enough  of  other  forms  are  left  to  spoil  the 
wine. 

A  " starter"  of  some  kind  is  therefore  necessary  with  defecated  must 
and  useful  in  all  other  cases. 

A  Starter. — One  method  of  producing  a  starter  is  to  gather  a  suitable  quantity 
of  the  cleanest  and  soundest  ripe  grapes  in  the  vineyard,  crush  them  carefully 
and  allow  them  to  undergo  spontaneous  fermentation.  Perfectly  ripe  grapes 
should  be  selected  and  the  fermentation  allowed  to  proceed  until  at  least  10 
per  cent  of  alcohol  is  produced.  If  imperfectly  ripened  grapes  are  used  or  the 
starter  used  too  soon,  the  principal  yeast  present  will  be  S.  apiculatus.  Toward 
the  end  of  the  fermentation,  S.  ellipsoideus  predominates.  From  4  to  12  1.  (i  to 
3  gallons)  of  this  starter  should  be  used  for  each  400  1.  (100  gallons)  of  grapes  or 
must  to  be  fermented.  Too  much  starter  should  not  be  used  in  hot  weather  or 
with  warm  grapes,  otherwise  it  may  be  impossible  to  control  the  temperature. 

This  starter  is  used  only  for  the  first  vat  or  cask.  Those  following  are  started 
from  the  first  fermentations,  care  being  taken  always  to  use  the  must  only  from  a 
tank  at  the  proper  stage  of  fermentation  and  to  avoid  all  tanks  that  show  any 
defect. 

An  improvement  on  a  natural  starter  of  this  kind  is  a  pure  culture  of  tested  yeast. 
Such  yeasts  are  being  used  extensively  in  most  wine-making  regions,  usually  with 
excellent  results.  The  methods  of  use  would  require  too  much  space  to  describe  here, 
but  they  are  simple  and  such  as  could  easily  be  devised  by  anyone  with  some  knowl- 
edge of  microbiological  technic.  They  do  not  aim  at  obtaining  an  absolutely  pure 
fermentation,  which  is  unnecessary,  but  endeavor  to  have  an  overwhelming  pro- 
portion of  a  thoroughly  tested  and  suitable  yeast  which  will  rapidly  and  perfectly 
attenuate  the  wine  before  the  few  injurious  microorganisms  present  have  time  to 
do  any  harm. 


WINE  615 

DURING  FERMENTATION. — However  carefully  the  injurious  germs 
have  been  excluded  and  the  good  yeast  increased,  fermentation  will  not 
be  successful  unless  conditions  as  favorable  to  the  latter  and  unfavorable 
to  the  former  as  possible  are  maintained. 

The  temperature  of  the  crushed  grapes  or  expressed  must  is  of 
importance.  If  it  is  below  15°,  unless  the  weather  is  warm,  the  grapes 
should  be  warmed  to  20°  or  25°.  Unless  this  is  done,  the  molds  and 
S.  apiculatus,  which  require  less  heat  than  S.  ellipsoideus,  will  develop 
more  quickly.  This  is  especially  true  when  starters  are  not  used.  In 
the  warmer  and  earlier  districts  the  grapes  are  practically  never  too 
cold.  On  the  other  hand,  unless  there  is  great  carelessness,  the  grapes 
are  never  too  warm  for  the  commencement  of  fermentation.  The 
warmer  they  are,  however,  the  more  artificial  cooling  will  be  necessary 
later,  and  the  sooner  it  will  have  to  be  applied. 

For  white  wine,  the  crushing  must  be  thorough  to  facilitate  the 
pressing  out  of  the  juice  which  is  fermented  alone.  For  red  wine,  it  is 
only  necessary  to  break  the  berries  as  the  fermentation  softens  the 
pulp  sufficiently  for  pressing.  If  the  grapes  are  not  crushed,  they 
ferment  unevenly  and  the  growth  of  injurious  mold  is  encouraged. 

The  must  should  be  thoroughly  saturated  with  air  at  the  beginning 
of  fermentation  to  insure  the  multiplication  of  the  yeast.  The  aeration 
received  in  the  processes  of  stemming,  crushing  and  pressing  is  usually 
sufficient  for  this  purpose.  More  aeration  would  be  harmful,  injur- 
ing the  flavor  and  color  of  the  wine  by  over-oxidation  and  promoting 
the  growth  of  injurious  aerobic  organisms.  An  objection  to  the  sterili- 
zation of  must  by  heat  is  the  expulsion  of  the  air  and  the  difficulty  of 
replacing  it  in  the  proper  amount. 

The  proper  use  of  sulphurous  acid  in  the  regulation  of  fermentation 
is  one  of  the  most  important  and  necessary  but  least  understood  parts 
of  the  wine-maker's  art.  Only  by  this  proper  use  can  wholesome 
wine  of  the  highest  quality  be  produced.  Improper  use  will  injure  or 
completely  spoil  the  wine.  Its  beneficial  effects  are  due  primarily  to 
its  action  on  microorganisms,  on  enzymes  and  on  the  color  of  the  wine. 

In  the  small  quantities  properly  used  in  winemaking,  it  is  antiseptic  in  a  degree 
varying  with  the  amount.  All  microorganisms  are  susceptible  to  its  action  in  vary- 
ing degrees.  Bacteria  are  particularly  sensitive,  molds  and  psuedo-yeasts  less  so, 
while  wine  yeast  is  the  most  resistant  of  the  ordinary  forms  found  in  must  and  wine. 

The  result  of  the  use  of  the  proper  amount  of  sulphurous  acid  in  crushed  grapes 


6l6  MICROBIOLOGY    OF    ALCOHOLIC    FERMENTATION 

and  must  before  fermentation  is  the  almost  complete  suppression  of  bacterial  action, 
the  discouragement  of  molds  and  pseudo-yeasts  and  the  promotion  of  the  growth 
of  wine  yeast  which  is  given  a  clear  field  unhindered  by  the  deleterious  excretions 
of  competitors. 

Its  action  as  regards  enzymes  is  hardly  less  important.  It  would  be  impossible  to 
make  the  finest  wines  of  Sauternes  and  the  Rheingau  without  its  use  on  account  of 
the  oxidase  produced  by  the  Botrytis  cinerea  which  is  abundant  and  necessary  on 
the  best  grapes  of  these  regions.  In  other  regions  where  this  mold  and  others 
occasionally  occur  its  use  is  also  necessary.  In  hot  climates  it  is  especially  useful, 
not  only  because  bacterial  action  is  more  intense  in  such  regions  but  because  of  its 
action  in  preserving  the  natural  fixed  acids  of  the  grape,  which  are,  there,  nearly 
always  deficient.  This  preservation,  according  to  Wortmann,  is  due  to  the  sup- 
pression of  acid-consuming  bacteria,  but  experiments  of  Astruc  tend  to  show  that 
the  prevention  of  the  action  of  unknown  acid-destroying  enzymes  is  in  part  the 
cause. 

Its  action  on  the  color  of  wines  is  also  of  importance.  By  the  action  of  oxygen, 
the  color  of  red  wine  is  gradually  made  insoluble  and  precipitated,  and  the  greenish 
or  golden  color  of  white  wine  is  turned  to  brown.  Both  these  actions  are  prevented 
or  much  diminished  by  the  use  of  minute  quantities  of  sulphurous  acid. 

The  most  commonly  used  source  of  sulphurous  acid  is  the  fumes  of  burning 
sulphur.  Sulphur  is  burned  in  a  cask  and  the  must  caused  to  take  up  the  fumes  by 
being  pumped  into  the  cask  through  the  upper  bung  hole.  It  is  almost  impracticable 
to  apply  sulphurous  acid  from  this  source  to  crushed  grapes  for  red  wine. 

The  method  is  defective  in  many  ways.  It  is  impossible  to  tell  within  very  wide 
limits  how  much  sulphur  dioxide  has  been  absorbed  by  the  wine.  Moreover,  the 
sulphur  burns  incompletely  and  the  volatilized  sulphur  acted  upon  by  the  yeast 
may  produce  sulphuretted  hydrogen.  Other  sulphur  compounds  are  also  pro- 
duced during  the  burning,  to  some  of  which  the  so-called  sulphur  taste  of  wine  is 
said  to  be  due.  Several  devices  have  been  invented  to  decrease  these  defects  but 
none  remove  them  completely;  accordingly  progressive  wine-makers  are  adopting 
more  reliable  sources. 

An  improvement  is  the  use  of  potassium  metabisulphite  (K2S2O5)  a  salt  which 
can  be  obtained  in  the  requisite  purity  in  commerce  containing  50  to  55  per  cent  by 
weight  of  sulphur  dioxide.  The  amount  of  potash  added  by  this  salt  in  the  doses 
used,  is  very  small,  and  far  within  the  limits  of  variation  between  different  wines. 
By  the  use  of  this  salt,  exact  amounts  of  sulphur  dioxide  can  be  applied  both  to 
white  and  red  wines.  Other  sulphites  are  not  permissible. 

The  best  source  of  the  acid,  recently  brought  into  limited  use,  is  the  liquefied  gas, 
which  can  be  manufactured  comparatively  cheaply  in  great  purity.  By  its  use  all 
the^benefits  of  sulphurous  acid  are  obtained  and  the  defects  eliminated. 

Some  grapes,  owing  to  their  composition,  especially  their  high 
acidity,  are  very  resistant  to  the  attacks  of  injurious  bacteria.  Others, 
owing  to  their  low  acidity  or  highly  nitrogenous  nature,  are  very 
susceptible.  The  addition  of  tartaric  or  citric  acid  to  the  latter  has 


WINE  617 


therefore  a  deterring  effect  on  some  of  the  most  dangerous  forms.  It 
is  seldom  necessary,  however,  to  modify  the  composition  for  this  pur- 
pose if  the  other  means  of  control  are  used.  The  addition  of  acid  or 
its  decrease  by  dilution  or  neutralization  should  be  solely  for  the  direct 
improvement  of  the  taste. 

The  quality  and  character  of  the  wine  depends  greatly  on  the  tem- 
perature of  fermentation.  If  too  low,  the  fermentation  may  be  unduly 
prolonged,  the  wine  yeast  may  have  difficulty  in  .overcoming  its  com- 
petitors and  the  wine  may  remain  inferior  and  cloudy.  With  red  wine, 
the  desired  color,  tannin  and  body  may  not  be  secured.  On  the  other 
hand,  if  the  temperature  is  too  high  the  results  are  worse.  The  growth 
of  bacteria  is  promoted,  injuring  the  wine  by  the  volatile  acid  and  dis- 
pleasing flavors  produced  and  preventing  the  proper  action  of  the  yeast. 
Such  wines  may  remain  sweet  on  account  of  the  failure  of  the  yeast  to 
do  its  work  and  become  unpleasantly  acid  owing  to  the  volatile  acids 
produced  by  the  bacteria. 

Some  means  of  controlling  the  temperature  is  therefore  always 
needed.  Where  heat  is  deficient  it  may  be  supplied  by  direct  heating 
of  the  must  or  part  of  it,  or  by  heating  the  cellar.  Where  the  heat  is 
excessive,  it  may  be  diminished  by  crushing  only  cold  grapes,  using 
small  fermenting  vats  to  promote  radiation  and  finally  by  the  use  of 
cooling  machines  applied  directly  to  the  fermenting  wine. 

The  best  temperature  for  fermentation  depends  on  the  kind  of  wine. 
For  light  white  wines,  the  maximum  should  not  exceed  25°,  for  heavier 
wines  30°,  while  for  heavy  red  wines  where  high  extract  and  tannin  are 
required,  it  may  be  allowed  to  reach  35°.  Sound  wines  can  be  made  at 
all  these  temperatures. 

As  already  explained,  the  ordinary  processes  of  treatment  of  grapes 
result  in  sufficient  aeration  for  the  multiplication  of  the  yeast.  With 
grapes  containing  little  sugar,  this  may  suffice  to  complete  fermentation. 
With  sweeter  grapes,  the  fermentation  usually  slackens  when  the 
alcohol  reaches  n  or  12  per  cent  by  volume  or  sooner,  unless  some 
supplementary  aeration  is  given.  With  white  wine  this  is  seldom  done, 
with  the  result  that  the  time  of  fermentation  is  prolonged.  With  red 
wine,  the  necessary  stirring  of  the  pomace  to  promote  color  extraction 
or  the  pumping  over  of  the  must  in  the  cooling  process  usually  gives  a 
large  amount  of  aeration  which  is  sometimes  excessive.  Too  much 
aeration  results  in  extremely  rapid  fermentation  and  consequent 


6l8  MICROBIOLOGY   OF   ALCOHOLIC   FERMENTATION 

difficulty  in  controlling  the  temperature.  It  may  also  have  a  dele- 
terious effect  on  the  color,  especially  if  sulphur  dioxide  has  not  been 
used. 

In  any  case,  the  main  part  of  the  fermentation  should  be  over  in 
from  three  to  five  days  in  the  case  of  red  and  in  from  seven  to  fourteen 
days  in  the  case  of  white  wine.  With  heavy  musts,  however,  there  will 
still  remain  from  0.5  to  i  or  2  per  cent  of  sugar.  With  certain  special 
wines  such  as  Sauternes  it  is  desirable  to  retain  the  slight  sweetness  due 
to  this  small  amount  of  unfermented  sugar.  This  is  accomplished  by 
the  judicious  use  of  sulphurous  acid,  prompt  clarification  by  filtration  or 
fining  and  when  necessary  by  pasteurization.  The  pasteurization  tends 
to  remove  those  proteins  which  are  coagulated  by  heat  and  which  are 
the  preferred  food  of  bacteria. 

In  the  case  of  dry  wines,  protection  from  bacteria  is  best  obtained 
by  prompt  and  complete  attenuation.  Fermentation  should  not  be  al- 
lowed to  cease  until  all  the  sugar  has  disappeared.  For  this  purpose, 
one,  two  or  more  aerations  by  pumping  over  are  usually  necessary  im- 
mediately after  the  end  of  the  tumultuous  fermentation.  The  tem- 
perature of  the  wine  should  not  be  allowed  to  fall  sufficiently  to  check 
the  action  of  the  yeast  until  all  the  sugar  has  disappeared. 

AFTER  FERMENTATION. — As  soon  as  all  the  sugar  has  been  destroyed 
in  the  case  of  dry  wines,  or  the  desired  degree  of  attenuation  has  been 
obtained  in  the  case  of  sweet  wines,  all  the  useful  work  of  microorgan- 
isms has  been  accomplished.  The  quality  and  safety  of  the  wine  then 
depend  on  freeing  it  from  all  organisms  present  and  preventing  the 
entrance  and  action  of  all  others. 

As  soon  as  bubbles  of  carbon  dioxide  cease  to  be  given  off,  the  yeast 
and  other  solid  matters  will  settle  to  the  bottom  and  the  liquid  become 
clear.  This  often  occurs  before  the  fermentation  is  complete.  In  this 
case  the  yeast  should  be  stimulated  by  aeration  as  described  above. 


If  the  wine  is  dry,  it  should  be  racked  (drawn  off,  decanted)  from  the  sediment 
into  clean  casks.  The  first  racking  is  usually  done  while  the  wine  is  still  slightly  cloudy 
during  the  first  month  or  six  weeks  to  remove  the  more  bulky  sediment.  If  left  too 
long  on  the  yeast  the  autophagy  or  degeneration  of  the  latter  may  produce  substances 
which  injure  the  brightness  and  flavor  of  the  wine. 

A  second  racking  is  necessary  at  the  end  of  winter  before  the  spring  rise  of  tem- 
perature tends  to  renew  the  activity  of  the  microorganisms  which  always  remain 
in  the  wine.  A  well  made  wine  at  this  time  should  be  perfectly  bright  and  all  solid 


WINE  619 

matters  consisting  of  yeast  and  bacteria,  coagulated  proteins  and  crystals  of  bi- 
tartrate  should  have  accumulated  in  the  sediment. 

Racking  should  take  place  when  possible  only  in  settled  weather,  when  the  baro- 
metric pressure  is  high.  Low  atmospheric  pressures  diminish  the  solubility  of  the 
carbon  doxide  with  which  the  wine  is  saturated.  Under  these  conditions,  therefore, 
bubbles  of  gas  are  apt  to  be  given  off,  bringing  up  particles  of  sediment  and  rendering 
the  wine  cloudy.  However  long  wine  is  kept  in  wooden  casks,  it  will  continue  to 
deposit  sediment  owing  to  chemical  changes  due  to  the  action  of  oxygen  which  pene- 
trates slowly  through  the  wood.  Repeated  rackings  are  therefore  necessary,  oc- 
curring at  least  twice  a  year  until  the  wine  is  bottled  or  consumed. 

Abundant  aeration  is  necessary  during  fermentation.  A  moderate  supply  of 
oxygen  is  necessary  for  the  proper  aging  of  wine.  Experience  has  shown  that  exactly 
the  proper  amount  of  pure  filtered  air  will  obtain  access  to  the  wine  for  the  latter  pur- 
pose through  the  wood  of  ordinary  casks  of  proper  size.  If  the  casks  are  too  small  the 
oxidation  may  be  too  rapid,  if  too  large  the  maturing  of  the  wine  may  be  unduly  pro- 
longed. The  temperature  of  the  storage  cellar  is  the  main  modifying  factor.  The 
warmer  the  cellar  the  larger  the  casks  should  be.  The  range  for  fine  wines  is  from 
5o-gallon  barrels  to  1000  gallon  casks.  Ordinary  wines  where  aging  is  unneces- 
sary or  impracticable,  may  be  stored  in  larger  containers. 

With  sound,  completely  fermented  wines,  all  aeration,  other  than  that  due  to  the 
porosity  of  the  wood,  should  be  avoided  as  much  as  possible.  This  is  accomplished 
by  keeping  the  casks  tightly  bunged  and  completely  filled.  Evaporation  through  the 
wood  continually  diminishes  the  volume  of  wine  and  the  lack  must  be  supplied  by 
filling  up,  at  first  two  or  three  times  a  month  and  later  every  month  or  two.  The 
drier  the  air  of  the  cellar,  the  more  frequent  the  fillings  necessary. 

A  light  sulphuring  of  the  clean  casks  into  which  the  wine  is  racked  is  usual. 
This  should  be  practised  with  great  caution.  Very  little  is  needed  with  sound  wines, 
especially  if  it  has  been  used  before  or  during  fermentation  and  a  slight  excess  will 
injure  the  flavor.  The  amount  should  not  exceed  i.25g.  per  hectoliter  for  red  or 
2  g.  for  white  wine.  One-half  to  one-third  of  this  is  sufficient  for  old  wines.  The 
amount  can  be  accurately  measured  only  when  using  metabisulphite  or  the  liquefied 
gas.  The  utility  of  the  sulphur  dioxide  with  perfectly  sound  wines  is  to  diminish 
oxidation;  with  wines  liable  to  disease,  to  discourage  the  growth  of  bacteria. 

All  manipulation  of  the  wine  should  be  conducted  with  strict  attention  to 
cleanliness.  This  applies  especially  to  empty  casks,  pumps  and  hoses.  These 
should  be  thoroughly  cleaned  immediately  after  use  and,  if  of  metal  or  non- 
absorbent  material,  kept  perfectly  dry.  Utensils  of  wood,  rubber  or  other  porous 
material  should  be  preserved  from  bacterial  or  mold  growth  with  sulphurous  acid. 

The  clarification  of  a  perfectly  sound  new  wine  may  be  facilitated  and  hastened 
by  thoroughly  stirring  up  the  yeast  one  or  two  days  before  racking.  The  yeast  in 
settling  carries  down  much  of  the  finer  suspended  matter,  thus  effecting  a  rough 
fining.  Materials  such  as  kaolin,  pure  silica  sand,  charcoal  and  filter-paper  can 
be  used  with  the  same  effect.  The  fining,  however,  is  never  perfect  and  the  flavor 
of  the  wine  is  often  injured.  A  very  pure  clay,  known  commercially  as  Spanish 
clay,  is  used  largely  for  clearing  sweet  wines  where  the  flavor  is  not  so  delicate. 
From  75  to  125  mg.  per  hectoliter  are  used  for  this  purpose. 


620  MICROBIOLOGY    OF    ALCOHOLIC    FERMENTATION 

The  best  wines  are  nearly  always  fined  at  least  once,  immediately  before  bottling. 
One  or  two  finings  may  precede  this  to  hasten  aging,  defecation  and  bottle  ripeness. 

The  materials  used  are  soluble  gelatinous  or  albuminous  substances  which  are 
capable  of  being  coagulated  and  precipitated  by  some  ingredient  of  the  wine.  The 
best  of  the  commonly  used  substances  are  isinglass  (ichthyocol)  2  or  3  g.  per  hecto- 
liter, for  white  wines;  the  white  of  fresh  eggs,  i  or  2  per  hectoliter  for  red;  and 
gelatin,  10  or  12  g.  per  hectoliter  for  either. 

The  proper  quantity  of  the  finings  is  dissolved  in  a  little  water  diluted  with  wine 
and  stirred  into  the  cask.  The  tannins  and  acids  of  the  wine  cause  a  gradual  coagula- 
tion in  minute  particles  throughout  the  liquid.  These  particles  gradually  coalesce, 
forming  larger  particles  which  include  all  the  other  floating  solid  matter  of  the  wine 
as  in  a  net.  These  larger  particles  contracted  by  the  alcohol  then  settle  to  the 
bottom,  leaving  the  wine  perfectly  bright. 

The  coagulum  consists  of  a  combination  of  the  gelatinous  matter  and  the  tannin. 
Some  of  the  latter,  therefore,  is  removed  from  the  wine.  With  astringent  red  wines, 
this  may  be  an  improvement.  If  there  is  no  excess  of  tannin  present,  enough  must 
be  added  to  combine  with  the  finings  used.  With  white  wines  which  contain  little 
or  no  tannin,  this  addition  is  always  necessary. 

The  amount  to  use  varies  with  the  quality  of  the  finings  and  of  the  tannin  and 
with  the  composition  and  temperature  of  the  wine. 

To  precipitate  commercial  gelatin  of  good  quality  about  an  equal  quantity  of 
good  tannin  is  necessary;  isinglass  properly  prepared  requires  only  from  one-half  to 
one-third  this  amount.  Eggs  require  only  minute  quantities. 

Specially  prepared  casein  of  milk  is  used  for  fining  white  wine.  Its  chief  merit  is 
that  the  acids  of  the  wine  alone  cause  its  complete  precipitation  and  no  addition  of 
tannin  is  needed,  though  a  little  is  sometimes  helpful.  Many  other  albuminous 
substances  such  as  milk,  blood  and  various  proprietary  preparations  are  also  used, 
but  they  are  all  inferior  to  the  three  mentioned  and  many  of  them  introduce  foreign 
matters  such  as  milk  sugar  and  bacteria  which  are  a  source  of  danger  to  the  wine. 

Wines  containing  many  disease-producing  bacteria  may  be  injured  by  the  intro- 
duction of  finings.  The  evolution  of  gases  due  to  the  bacterial  action  may  prevent 
the  settling  and  the  protein  matters  introduced  will  favor  the  multiplication  of  the 
disease-producing  organisms.  By  the  use  of  5  to  10  g.  of  sulphurous  acid  per 
hectoliter  added  to  the  wine  immediately  before  the  addition  of  the  gelatin,  the 
bacteria  may  be  temporarily  paralyzed  and  the  finings  will  then  settle  and  remove 
the  bacteria  with  the  other  floating  particles. 

The  bright  wine  should  be  racked  from  the  finings  very  soon  after  the  sediment 
has  settled,  especially  when  disease-producing  bacteria  are  numerous.  This  will 
be  in  from  ten  to  twenty  days.  If  the  wine  is  not  clear  in  three  weeks  it  should  be 
filtered. 

Filtering  is  inferior  to  fining  in  producing  a  perfectly  bright  wine.  It  is  more 
rapid,  however,  and  is  useful  in  clearing  common  wine  and  wines  refractory  to  fining. 

Filters  of  innumerable  forms  are  used.  They  are  of  two  main  types.  For  rough 
clearing  of  very  cloudy  wines  some  form  x)f  bag  filter  is  usually  employed  in  which  the 
wine  passes  through  a  cloth  tissue.  The  passage  at  first  is  rapid  and  the  nitration 
imperfect.  As  the  solid  matter  accumulates  on  the  filtering  surface,  the  filtration  im- 


WINE  621 

proves  but  the  passage  of  the  wine  is  retarded.  The  first  wine  is  passed  a  second  time 
through  the  filter  and  as  soon  as  the  rate  of  nitration  becomes  too  slow,  the  operation 
must  be  stopped  and  the  filtering  surface  renewed. 

For  wines  containing  little  sediment,  the  filter  must  be  primed.  This  is  accom- 
plished by  putting  some  finings  in  the  wine  first  passed  through  the  filter.  The 
priming  is  more  effective  and  the  output  of  the  filter  much  increased  if  a  little  in- 
fusorial earth  is  used  with  the  gelatin. 

For  the  more  perfect  clearing  of  old  wines  some  form  of  pulp  filter  is  used.  These 
are  various  devices  by  which  the  wine  is  forced  through  a  mass  of  cellulose  or  as- 
bestos pulp  and  freed  from  all  floating  matter.  Some  of  the  best  of  these,  carefully 
used,  remove  nearly  all  of  the  bacteria  present. 

PROHIBITION  AND  WINE. — Legislation  restricting  or  prohibiting 
the  manufacture,  sale  or  use  of  alcoholic  beverages  has  profoundly 
affected  the  industry,  especially  in  the  United  States,  where  the 
making  of  wine  and  other  alcoholic  beverages  on  a  commercial  scale 
is  now  illegal  except  for  sacramental  and  other  specified  purposes. 
Whether  it  will  be  permitted  as  a  home  industry  for  family  use  is  still 
uncertain. 

Other  uses  for  wine-grapes  are  therefore  being  sought.  In  the 
eastern  states  many  can  be  used  for  the  manufacture  of  grape  juice.  In 
California  the  most  promising  means  of  profitably  disposing  of  the  crop 
are  drying  and  the  manufacture  of  grape  syrup.  These  products, 
especially  the  latter,  will  have  some  value  as  foods  but  their  main  outlet 
must  be,  at  first,  in  those  countries  where  wine-making  is  permitted. 

Wine  and  vinegar  can  be  made  from  both  of  these  products  by  essen- 
tially the  same  methods  described  for  fresh  grapes.  The  dilution  of 
the  syrup  and  the  extraction  of  a  must  of  suitable  concentration  from 
the  dried  grapes  offer  slight  mechanical  difficulties  which  can  easily  be 
overcome.  The  use  of  starters  of  selected  yeast  will  be  necessary  and 
will  offer  some  difficulty  in  operation  on  a  small  scale. 

The  quality  of  the  wine  will  be  inferior  in  some  cases  and  fair  to 
good  in  others.  The  color  of  red  grapes  is  almost  entirely  destroyed  by 
sun-drying  but  remains  in  grapes  dried  in  evaporators.  The  aromas 
and  flavors  of  the  grapes  are  modified  also  more  by  sun  drying  and 
concentration.  The  effects  of  these  modifications  in  the  resulting  wine 
are  both  favorable  and  unfavorable.  The  wines  will  have  less  marked 
aromas  but  will  age  more  quickly.  The  wines  made  from  dried  grapes 
will  tend  to  be  high  in  tannin  and  extract;  those  made  from  grape  syrup, 
low  in  acid  and  extract.  A  combination  of  the  two  raw  materials 
will  probably  give  the  best  results. 


CHAPTER  II 


BEER 

Beer  is  an  alcoholic  beverage  made  from  certain  cereal  grains  by 
transformation  of  the  starch  to  sugar,  dilution  with  water,  and  fermen- 
tation with  yeast.  There  is  usually  an  addition  of  hops  and  sometimes 
of  materials  containing  sugar.  The  liquid  before  fermentation 
called  wort. 

TYPICAL  COMPOSITION  OF  VARIOUS  BEERS 


is 


Lager 

Ale 

Porter 

Weisbier 

Temper- 
ance beer 

Water  

•4 

GO    4O 

88   30 

8?    3O 

02  oo 

Alcohol  (by  vol.)  

4.85' 

8.00 

7  .00 

7     At; 

2   OO 

Extract  

4.  2O 

ir    CA 

6  4^ 

4    63 

3O<» 

Sugar 

i  60 

I    33 

I    83 

I    71 

I    08 

Lactic  acid  

i 

O    IO 

O    2O 

O    22 

O    27 

O    O4 

Ash  

o.  23 

O.  3O 

o  40 

o  16 

RAW  MATERIALS  AND  MICROORGANISMS  OF  BREWING 
GRAINS  EMPLOYED. — Barley,  rice  and  maize  are  the  grains  most  commonly  used, 
wheat,  rye  and  oats  but  rarely.     Cane  and  beet  sugars  and  syrups  sometimes  form 
part  of  the  fermentable  material. 

YEASTS  or  BEER. — The  yeast  used  is  usually  one  of  the  many  forms 
of  5.  ceremsia.  In  some  spontaneously  fermented  beer,  other  yeasts, 
Torulce,  and  bacteria  take  part,  but  in  ordinary  beers  most  of  these  are 
considered  as  disease-producing  organisms  and  injurious. 

KINDS  OF  BEER. — The  principal  varieties  of  beer  are:  lager  beers,  fermented  with 
bottom  yeasts;  ales,  fermented  with  top  yeasts  (and  Torul(R)\  porters,  similar  to  ale 
but  dark  in  color  owing  to  the  use  of  caramelized  malt;  weisbiers,  in  which  lactic 
bacteria  are  abundant;  and  certain  local  types  in  which  bacteria  produce  con- 
siderable quantities  of  lactic  and  acetic  acids.  Many  attempts  have  been  made  to 
produce  a  beverage  similar  to  beer  but  lacking  the  alcohol.  The  ''temperance 
beers"  are  of  this  character.  Some  of  them  are  made  by  restricting  the  fermen- 
tation or  by  omitting  it.  These  are  little  more  than  wort  or  decoctions  of  malt 
and  lack  the  products  of  fermentation  to  which  much  of  the  flavor  of  beer  is  due. 
In  others  the  beer  is  made  in  the  usual  way  and  then  by  various  devices  freed 
from  alcohol.  The  latter  are  more  expensive  to  produce  but  are  considered  of 
superior  quality. 

622 


BEER  623 

PROCESS  OF  BREWING 

OUTLINE. — The  manufacture  of  beer  takes  place  in  four  main  stages.  First, 
a  portion  or  all  of  the  grain  is  soaked  in  water,  allowed  to  germinate  and  then 
dried.  This  produces  the  malt  which  contains  the  enzymes  necessary  for  the 
conversion  of  the  starch  into  sugar  and  the  disintegration  of  the  tissues  of  the  grain. 
The  malt  is  then  crushed  (and  usually  mixed  with  unmalted  cereals  or  sugar)  and 
heated  with  water.  This  constitutes  mashing.  During  this  process,  the  starch 
changes  to  maltose  and  dextrins  which  with  other  matters  dissolve  in  the  water; 
then  bacteria  produce  a  small  amount  of  lactic  acid.  The  resulting  solution  con- 
stitutes the  wort. 

The  wort,  by  the  addition  of  yeast  is  fermented  and  changed  to  beer.  The 
fourth  stage  includes  all  manipulation  of  the  fermented  beer  to  prepare  it  for  con- 
sumption. 

MALTING:  PRODUCTION  OF  ENZYMES. — The  best  malt  is  made  from 
barley,  but  for  special  beers  may  be  made  from  wheat  or  other  grains. 
Steeping  consists  in  soaking  in  water  to  start  germination.  This 
requires  from  thirty-six  to  seventy-two  hours  and  causes  an  increase  in 
weight  of  about  45  per  cent.  The  temperature  should  be  about  12.5°. 
If  higher,  injurious  molds  will  develop.  If  much  lower,  germination 
will  be  retarded.  The  water  should  contain  little  organic  matter  or 
chlorides,  nitrates  or  iron  salts.  A  little  calcium  sulphate  is  favor- 
able. If  it  contains  many  microorganisms  it  should  be  sterilized  by 
boiling.  A  very  little  sulphite  of  lime  or  of  potassium  may  be  used  to 
discourage  molds. 

During  germination  several  enzymes  appear,  of  which  the  most  important  to  the 
brewer  are  diastase  which  changes  insoluble  starch  into  soluble  sugar,  rendering  it 
available  for  the  growth  of  the  young  plant;  peptase,  which  performs  a  similar 
function  as  regards  nitrogenous  matters;  and  cytase  which  helps  in  the  disintegration 
of  the  cellulose.  All  these  are  necessary  to  prepare  for  the  work  of  the  yeast. 
When  the  plumule  has  grown  to  about  two-thirds  the  length  of  the  grain,  sufficient 
enzymes  have  been  formed.  This  requires  from  about  sixteen  to  twenty  days. 

The  growth  of  the  sprouting  seed  is  at  this  point  stopped  by  careful  drying  with 
artificial  heat  in  a  kiln.  The  kilning  must  be  sufficiently  rapid  to  kill  the  germinat- 
ing seedling  quickly,  but  not  too  rapid  or  at  too  high  a  temperature,  otherwise  the 
enzymes  will  be  weakened  or  destroyed.  The  enzymes  are  more  sensitive  when 
moist,  consequently  the  heat  may  be  increased  as  drying  proceeds.  The  process 
commencing  at  a  temperature  between  30°  and  35°  is  increased  gradually  to 
50°  and  55°.  In  twelve  to  twenty-four  hours,  the  malt  should  appear  dry.  The 
temperature  is  again  raised  gradually  for  another  twelve  to  twenty-four  hours  to 
8o°-ioo°.  The  lower  the  temperature  the  lighter  the  color  of  the  malt.  Higher 
temperatures,  especially  while  the  malt  is  moist,  produce  dark  malt. 


624  MICROBIOLOGY   OF  ALCOHOLIC   FERMENTATION 

As  soon  as  the  kilning  is  finished  the  radicles  are  removed  by  friction  and  screening 
in  special  machines. 

WORK  OF  ENZYMES  AND  BACTERIA. — The  malt  is  first  crushed  by 
pressing  between  rollers  to  facilitate  the  work  of  the  enzymes  and  the 
solvent  action  of  the  water.  If  unmalted  grain  is  to  be  used  as  well,  this 
must  be  ground  and  the  starch  made  soluble  by  heating  under  pressure 
with  three  or  four  times  its  weight  of  water  and  a  little  malt  to  8o°-85° 
for  about  an  hour. 

The  methods  of  mashing  are  very  various.  They  consist  in  general  of  mixing  the 
ground  malt  with  warm  water,  bringing  the  mass  to  a  temperature  of  35°  to  45°  which 
is  gradually  raised  to  6o°-65°  by  the  addition  of  hotter  water.  When  the  action  of 
the  enzymes  commences,  the  heated  decoction  of  unmalted  grains  is  added  in  va- 
rious ways,  and  the  temperature  controlled  by  additions  of  hot  water  or  by  heating 
a  portion  of  the  mash.  The  whole  mashing  process  requires  from  two  to  five  hours 
according  to  the  methods  used. 

During  the  mashing,  the  starch  is  transformed  partly  into  maltose  and  partly  into 
dextrins.  The  ratio  of  these  products  will  vary  according  to  the  amount  of  diastase 
present  and  especially  according  to  the  temperature  used.  At  about  60°  the  maxi- 
mum amount  of  maltose  is  produced;  at  higher  temperature  (65°  to  75°)  the 
unfermentable  dextrins  increase.  The  amount  of  alcohol  and  the  amount  of  extract 
in  the  beer  therefore  depend,  to  a  great  extent  on  the  method  of  mashing. 

During  the  first  part  of  the  mashing,  while  the  temperature  is  about 
45°,  lactic  bacteria  develop.  If  their  action  is  too  intense  they  will 
render  the  beer  unpleasantly  acid.  If  moderate,  the  acidity  they  com- 
municate to  the  wort  is  useful  in  preventing  the  growth  of  the  harmful 
butyric  bacteria  which  might  develop. 

After  mashing,  the  wort  is  separated  from  the  solid  matters  by  drawing  off, 
extracting  the  mash  with  hot  water  (sparging),  and  filtration.  It  is  then  boiled  from 
one  to  eight  hours  according  to  the  result  desired. 

Boiling  sterilizes  the  wort,  kills  all  bacteria  and  destroys  any 
enzymes  which  remain.  These  results  are  obtained  almost  instantane- 
ously owing  to  the  lactic  acid  present.  Coagulation  of  protein  sub- 
stances is  also  brought  about,  effecting  a  clarification  of  the  wort. 
This  requires  one  or  more  hours,  according  to  the  nature  of  the  wort. 
It  is  necessary  also  in  some  cases  to  concentrate  the  wort,  which  is 
done  by  prolonged  heating  in  open  kettles.  This  may  require  several 
hours. 

The  Hopping  of  the  wort  takes  place  during  the  boiling.  Sometimes 
the  hops  are  added  just  at  the  end  of  boiling;  sometimes  in  two  or  three 


BEER  625 

portions,  one  of  which  may  be  at  the  beginning  and  one  after  boiling. 
Hops  contain  an  aromatic  essential  oil,  resins  and  tannin.  The  essential 
oil  is  quickly  soluble  and  volatile.  To  preserve  its  aroma  in  the  beer, 
the  hops  must  not  be  boiled  too  long.  The  resins  are  antiseptic  and 
help  to  preserve  the  beer.  They  dissolve  with  more  difficulty  and 
require  longer  boiling. 

FERMENTATION:  WORK  OF  YEAST. — After  boiling,  the  wort  is  sepa- 
rated from  the  hop  debris  by  straining.  It  is  then  cooled  by  means  of 
refrigerators  consisting  usually  of  serpentine  tubes  through  which  cold 
brine  or  water  runs.  The  hot  wort  runs  or  drips  over  the  outside  of 
these  tubes  in  contact  with  the  air.  The  final  temperature  of  the  wort 
is  from  12°  to  1 8°  in  top  fermentation  and  4°  to  6°  in  bottom. 

By  this  means  the  wort  is  thoroughly  aerated,  which  is  necessary  for 
the  proper  work  of  the  yeast.  It  also  effects  a  partial  clarification  by 
oxidation  which  causes  a  precipitation  of  solid  matters. 

The  fermentation  takes  place  in  two  stages,  the  violent  or  tumultu- 
ous fermentation  in  vats  and  the  secondary  or  after  fermentation  in 
casks. 

During  the  violent  fermentation,  the  temperature  is  allowed  to 
reach  a  maximum  of  7°  to  9°  with  light  beers,  8.5°  to  10.5°  with  dark  and 
12°  to  20°  in  top  fermentations.  At  the  end  of  the  first  fermentation, 
the  beer  is  cooled  gradually  to  3.5°  or  5.0°  and  drawn  into  fermenting 
casks  where  the  after-fermentation  takes  place. 

The  yeasts  used  in  brewing  vary  very  much.  Besides  the  division 
into  top  and  bottom  yeasts,  various  types  of  each  are  recognized.  One 
of  the  chief  characteristics  used  for  this  division  is  expressed  by  the  per- 
centage of  the  total  extract  fermented  by  the  yeast.  The  Saaz  type 
leaves  all  the  dextrins  and  some  of  the  maltose  untouched  and  produces 
beers  light  in  alcohol  and  high  in  extract.  The  Logos  type  destroys  all 
the  maltose  and  much  of  the  dextrins.  The  result  is  high  alcohol  and 
low  extract.  The  Frohberg  type  is  intermediate.  These  differences 
are  probably  due  to  differences  in  the  amount  and  perhaps  in  the 
kinds  of  enzymes. 

The  yeasts  of  spontaneously  fermenting  beers  are  of  various  species, 
S.  ellipsoideus,  S.  pasteurianus  and  others. 

To  produce  fermentation,  yeast  is  taken  from  previous  vats  so  long 
as  the  yeast  remains  sufficiently  uncontaminated  with  foreign  organisms. 
The  condition  of  the  yeast  is  determined  by  the  character  of  the  f  ermen- 

40 


626  MICROBIOLOGY   OF  ALCOHOLIC  FERMENTATION 

tation,  the  degree  of  attenuation,  and  by  microscopic  examination.  In 
breweries  where  modern  pure  culture  methods  are  not  used,  the  yeast 
present  is  always  of  several  forms  or  types. 

In  any  case,  after  a  certain  number  of  transfers,  the  yeast  deteriorates 
and  finally  may  become  thoroughly  infected  with  bacteria.  The  bac- 
teria are  revealed  by  microscopic  examination.  Where  pure  cultures 
are  used,  contamination  with  foreign  yeast  is  shown  by  a  change  in  the 
time  of  spore  formation.  By  this  method  a  contamination  of  1:200 
may  be  discovered. 

When  the  yeast  becomes  contaminated,  a  new  start  must  be  made 
with  yeast  from  another  brewery,  which  is  uncertain  or  by  a  starter  of 
pure  yeast,  which  is  the  only  reliable  method. 

The  new  start  with  pure  yeast  may  be  made  by  employing  a  kilo- 
gram of  pure  pressed  yeast  or  a  corresponding  amount  of  liquid  yeast 
and  gradually  increasing  it  to  the  desired  amount  by  repeated  small 
additions  of  sterile  wort.  This  must  be  done  with  special  precautions 
against  contamination.  Many  large  breweries  use  large  pure  yeast 
machines  which  produce  directly  sufficient  yeast  to  start  a  fermenting 
vat. 

AFTER  TREATMENT. — The  violent  fermentation  requires  from  eight 
to  eighteen  days  according  to  the  temperature.  It  takes  place  in  open 
vats  or  sometimes,  in  top  fermentation,  in  barrels.  When  sufficiently 
attenuated,  the  beer  is  drawn  off  into  large  casks  where  the  slow 
secondary  fermentation  takes  place  at  a  low  temperature  and  the  beer 
clears  by  depositing  yeast  and  other  sediment.  The  time  required  for 
the  secondary  fermentation  is  from  six  to  ten  weeks  or,  with  certain 
types  of  beer,  from  two  to  four  months  or  longer. 

A  certain  amount  of  dissolved  carbonic  acid  is  necessary  for  the 
quality  and  keeping  of  the  beer.  This  is  obtained  by  tightly  bunging 
the  casks  at  a  suitable  stage  of  the  secondary  fermentation. 

The  clarification  of  the  beer  is  sometimes  assisted  by  placing  a  quantity  of  chips 
of  beech  or  other  tasteless  wood  in  the  casks.  Top  fermentation  beers  are  often 
fined  by  the  use  of  isinglass  or  animal  gelatin.  Low  fermentation  beers  are  usually 
filtered. 

The  beer  is  then  ready  for  delivery  to  the  consumer  and  is  placed  in  barrels  with 
precautions  to  retain  the  dissolved  carbonic  acid. 

The  clear  beer  may  be  put  directly  into  bottles  with  the  same  precautions.  Bot- 
tled beers  which  are  to  be  kept  for  some  time  or  which  are  to  be  shipped  to  a  dis- 
tance are  pasteurized  after  bottling  at  60°  to  65°. 


BEER  627 

DISEASES  OF  BEER 

Beer  may  show  defects  due  to  imperfections  in  the  raw  material  or 
in  the  methods  of  manufacture.  These  are  principally  abnormal  flavors 
and  lack  of  clearness. 

The  diseases  properly  so  called  are  due  to  wild  yeasts  or  to  bacteria. 
The  disease-producing  yeasts  may  be  derived  from  the  starter,  from  the 
vessels  with  which  the  beer  comes  in  contact,  or  from  the  air.  They 
develop  most  commonly  during  the  secondary  fermentation  or  in  the 
bottle.  Some  may  produce  a  disagreeable  bitterness  (S.  pasteurianus 
I)  or  other  unpleasant  flavor  (S.  fcetidus) ;  many  produce  a  persistent 
cloudiness  (5.  ellipsoideus,  S.  apiculatus,  S,  exiguus,  S.  anomalus). 
They  are  to  be  combated  by  preventing  contamination,  by  proper 
attenuation  and  by  pasteurizing. 

Bacterial  diseases  were  more  common  before  effective  methods  of 
purifying  yeasts  were  known. 

Many  forms  of  lactic  bacteria  may  affect  the  beer,  rendering  it  acid 
and  cloudy.  They  occur  principally  where  the  temperature  is  allowed 
to  become  too  high  and  where  proper  care  in  the  cleaning  and  steril- 
ization of  utensils  is  not  exercised. 

Acetic  bacteria  may  occur  under  the  same  conditions  and  give  a 
taste  of  vinegar  to  the  beer.  They  are  more  common  in  top  fermented 
beers. 

Various  forms  of  Sarciniz  may  cause  persistent  cloudiness,  acid, 
unpleasant  flavors  or  both.  This  contamination  may  be  from  the  air 
or  the  water  and  is  relatively  common.  Their  growth  is  most  rapid 
at  1 6°  to  20°  and  is  retarded  by  the  antiseptic  properties  of  hops. 

Several  kinds  of  bacteria,  bacilli,  cocci  and  sarcinae  may  cause  the 
beer  to  become  slimy  or  viscid  and  injure  the  flavor.  This  trouble  is 
particularly  common  in  spontaneously  fermented  beer. 

Wort  and  beer,  being  organic  solutions  containing  very  little  acidity, 
are  favorable  media  for  the  growth  of  bacteria,  many  forms  of  which 
may  cause  trouble.  With  modern  methods  of  using  pure  yeast, 
cleanliness  and  the  pasteurization  of  bottled  beer,  diseases  can  be 
controlled. 


CHAPTER  III 
MISCELLANEOUS  ALCOHOLIC  BEVERAGES  AND  PRODUCTS 

CIDER  AND  PERRY 

These  beverages  are  made  by  the  alcoholic  fermentation  of  the 
juices  of  apples  and  pears  respectively  and  come  next  to  wine  and  beer 
in  the  quantities  produced. 

The  composition  of  the  fruit  varies  very  much  according  to  the 
variety,  especially  in  the  matters  of  acidity,  tannin  and  pectic  sub- 
stances. The  following  analysis  is  that  of  a  good  cider  apple: 

Sugar 167.0  g.  per  liter. 

Tannin 2 . 4  g.  per  liter. 

Acidity  (as  sulphuric) i .  6  g.  per  liter. 

The  pectic  matters  vary  from  2  g.  to  25  g.  per  liter  but  should  not 
be  too  high.  Pears  contain  usually  about  the  same  amount  of  sugar 
as  apples,  more  tannin  and  much  less  pectic  substances. 

The  microorganisms  occurring  naturally  on  the  surface  of  the  fruit 
are  similar  to  those  occurring  on  grapes,  but  special  forms  of  Sac- 
charomyces  are  found.  Pure  cultures  of  wine  yeast  are  used  success- 
fully in  cider  making  where  a  perfectly  dry  cider  is  wanted.  Where  a 
small  remnant  of  unfermented  sugar  is  desired,  the  difficulties  of  using 
pure  cultures  have  not  yet  been  overcome.  The  wild  yeasts  occurring 
on  the  fruit  in  large  quantities  usually  take  precedence. 

Attempts  to  sterilize  the  juice  by  heating  have  not  been  successful 
owing  to  the  production  of  a  persistent  cloudiness.  Sulphurous  acid 
is  even  more  effective  than  in  grape  juice  in  delaying  or  preventing  the 
action  of  the  microorganisms.  Its  use  must  therefore  always  be 
supplemented  by  a  starter  of  pure  yeast. 

The  principles  of  the  control  of  the  microorganisms,  good  and 
bad,  are  the  same  as  in  wine  making.  The  same  care  in  gathering 
and  keeping  the  fruit  and  in  extracting  and  handling  the  juice  are 
necessary. 

628 


MISCELLANEOUS  ALCOHOLIC  BEVERAGES  AND  PRODUCTS  629 

The  fermentation  is  similar  to  that  of  wine,  but  the  cider  should  be 
taken  off  the  yeast  sooner  in  order  to  promote  clarification  and  the 
retention  of  a  little  unfermented  sugar. 

Cider  is  subject  to  the  same  bacterial  alterations  as  wine  and  requires 
the  same  treatment.  It  is  more  difficult  to  keep  when  made  in  the 
ordinary  way  and  is  usually  consumed  during  the  first  year.  It  is 
particularly  subject  to  turning  brown,  owing  to  the  large  amount  of 
oxidase  present  in  apple  juice. 

The  use  of  sulphurous  acid  for  preliminary  defecation,  pure  yeast  in 
the  fermentation,  and  fining,  followed  by  pasteurization  soon  after  the 
fermentation,  seem  to  offer  the  best  means  of  improving  present 
methods. 

These  methods  were  introduced  into  a  cider-vinegar  factory  in 
California  by  W.  V.  Cruess  with  excellent  results. 

FERMENTED  BEVERAGES  OF  VARIOUS  FRUITS 

Many  other  fruits,  especially  those  rich  in  sugar  and  with  moderate 
acidity,  are  used  locally  to  produce  alcoholic  beverages.  The  methods 
of  fermentation  are  similar  to  those  used  in  wine  making,  but  additions 
of  sugar  and  water  are  usually  made  to  correct  defects  of  composition. 
Very  often  distilled  alcohol  is  also  added  after  fermentation  to  preserve 
the  liquid,  which  is  thus  rendered  unsuitable  for  an  ordinary  beverage. 

HYDROMEL  OR  MEAD 

An  alcoholic  beverage  made  by  the  fermentation  of  honey  and  water 
is  much  used  in  eastern  Europe. 

Honey  contains  from  65  to  74  per  cent  of  reducing  sugars  and  from 
2  to  10  per  cent  of  saccharose.  It  is  diluted  with  water  to  reduce  its 
concentration  to  22°  Bal.*-24°  Bal.  A  few  yeast  cells  are  usually 
present  in  the  honey  but  these  are  of  various  kinds  and  often  unsuit- 
able. The  use  of  a  good  pure  yeast  is  therefore  advisable.  As  honey 
contains  little  mineral  or  nitrogenous  yeast  food,  an  addition  of  nutritive 
substances  is  often  necessary: 

•  "Balling"  refers  to  the  degrees  of  the  special  hydrometer  for  determining  the  specific 
gravity  of  saccharine  solutions  such  as  must  or  beer  wort.  Its  purpose  is  to  indicate  directly 
the  percentage  of  solids  in  solution  at  a  temperature  of  6o°F. 


630  MICROBIOLOGY  OF  ALCOHOLIC  FERMENTATION 

The  following  formulae  are  recommended  by  Kayser  and  Boullanger 
to  be  used  in  one  liter: 

A.  Dicalcic  phosphate i      g. 

Ammonia 2      g. 

Bitartrate  of  potash 2      g. 

Magnesium  sulphate o .  i  g. 

B.  Maltopeptone i .  5  g. 

Bitartrate  of  potash i .  5  g. 

Ammonium  phosphate i .  o  g. 

The  same  results  may  be  obtained  by  mixing  from  20  to  50  per  cent, 
of  the  juice  of  grapes,  apples,  or  other  acid  fruits  with  the  diluted  honey. 

MISCELLANEOUS  FERMENTED  BEVERAGES 

Fermented  beverages  of  some  kind  are  made  in  practically  every 
part  of  the  world.  They  are  very  numerous  and  varied  but  fall  natur- 
ally into  three  groups;  those  made  from  the  sweet  juices  of  fruits  or 
other  plants  in  which  the  methods  of  manufacture  resemble  those  of 
wine  making;  those  made  from  starchy  materials  in  which  the  methods 
resemble  those  of  brewing;  and  finally  those  made  from  the  milk  of 
cows  or  other  mammals  which  are  discussed  in  Chapter  IV,  Div.  IV. 

Belonging  to  the  first  group  are  numerous  beverages  made  from 
the  juices  of  sugar  cane,  various  palms,  and  tropical  fruits.  The  best 
known  of  these  is  the  MEXICAN  PULQUE  made  by  the  spontaneous 
fermentation  of  the  sweet  juice  of  the  agave.  Little  is  known  about  the 
microflora  concerned,  but  it  includes  alcohol-forming  organisms  which 
produce  about  6  per  cent  of  alcohol,  and  bacteria  which  cause  rapid 
deterioration  and  spoiling  of  the  fermented  product.  The  pulque  is 
ready  for  consumption  twenty-four  hours  after  the  commencement  of 
fermentation  and  cannot  be  kept  more  than  a  day  or  two. 

Of  the  beverages  produced  from  starchy  materials  the  Japanese  SAKE, 
RICE  BEER,  has  been  most  studied.  It  is  made  from  rice  by  the 
diastatic  action  of  Aspergillus  oryz<z  and  yeast  fermentation.  The 
process  includes  three  stages.  First  the  preparation  of  koji  which 
consists  of  steamed  rice  on  which  the  spores  of  the  fungus  are  sown 
and  allowed  to  grow  at  20°  until  the  whole  mass  is  penetrated  with 
mycelium.  The  next  stage  is  the  preparation  of  moto  which  is  a  thick 
liquid  consisting  of  steamed  rice,  water  and  koji  in  which  the  fungus 
transforms  the  starch  into  sugar  at  o°  to  10°  in  a  few  days.  Fermen- 


MISCELLANEOUS  ALCOHOLIC  BEVERAGES  AND  PRODUCTS  631 

tation  then  starts  spontaneously,  alcohol  being  produced  by  the  action 
of  several  yeasts  and  lactic  acid  by  bacteria,  both  present  accidentally. 
In  about  two  weeks  the  moto  is  ready.  The  last  stage  is  the  principal 
fermentation  which  occurs  on  mixing  together  steamed  rice,  koji,  moto 
and  water.  This  requires  two  weeks.  The  liquid  is  then  separated, 
cleared  and  stored.  It  contains  a  considerable  amount  of  alcohol  and 
can  be  kept  and  aged  like  wine.  Sake  is  said  to  average  18  per  cent 
of  alcohol  and  may  reach  24  per  cent,  the  highest  alcohol  content 
known  to  be  produced  by  fermentation. 

POMBE  is  a  kind  of  beer  made  in  Africa  from  millet  seed  by  sprouting 
to  saccharify  the  starch  and  subsequent  spontaneous  fermentation  in 
water.  It  is  interesting  as  the  source  of  the  genus  Schizosaccharomyces 
which  appears  to  take  the  main  part  in  the  fermentation. 

GINGER  BEER  is  an  acid,  slightly  alcoholic  beverage  made  by  the 
fermentation  of  a  10  to  20  per  cent  solution  of  sugar  containing  a  few 
pieces  of  ginger  root.  The  fermentation  is  induced  by  adding  small 
pieces  of  the  so-called  ginger-beer  plant  which  consists  of  Bad.  vermi- 
jorme  and  S.  pyriformis.  The  bacteria  form  a  thick  gelatinous  sheath 
and  seem  to  live  symbiotically  with  the  yeast,  each  developing  best 
in  the  presence  of  the  other. 

DISTILLED  ALCOHOL 

INTRODUCTION 

USES  AND  SOURCES  OF  ALCOHOL. — Distilled  alcohol  is  used  as  a 
beverage  and  a  medicine  or  for  innumerable  purposes  in  the  arts  and 
industries.  Certain  methods  and  sources  employed  for  the  latter  pur- 
poses are  inadmissible  for  the  former. 

In  all  cases,  it  is  made  by  the  preparation  from  saccharine  or  starchy 
substances  of  a  sugar  solution  suitable  for  the  work  of  yeast,  the 
fermentation  of  this  solution,  and,  finally,  the  distillation  of  the  alcoholic 
liquid. 

Where  the  raw  materials  are  sugary,  methods  similar  to  those  of 
wine-making,  and  where  starchy,  to  those  of  brewing,  are  employed, 
modified  to  suit  the  conditions  of  each  case. 

The  principal  potable  alcohols  are  brandy,  made  from  grapes,  rum 
from  sugar  cane,  and  whiskey  from  rye  or  other  grains.  Many  other 


632  MICROBIOLOGY   OF   ALCOHOLIC   FERMENTATION 

sources  are  used  and  any  fermented  beverage  will,  by  distillation 
produce  a  potable  spirit  varying  in  character  and  quality  with  the 
source.  Industrial  alcohol  may  be  made  from  any  substance  capable  of 
undergoing  alcoholic  fermentation,  the  limiting  factor  in  practice  being, 
principally,  the  cost  of  the  raw  material  per  unit  of  alcohol. 

METHODS 

PREPARATION  OF  THE  SUGAR  SOLUTION. — Saccharine  Raw  Materials. 
When  spirits  are  to  be  made  from  grapes  or  other  fruit,  the  juice  is  fer- 
mented in  the  same  way  as  for  the  corresponding  beverage  and  then 
distilled.  The  juice,  however,  is  diluted  to  20°  Bal.  or  less,  as  it  is  not 
necessary  or  desirable  to  have  too  much  alcohol  in  the  fermented 
liquid.  The  product  is  consumed  directly  as  brandy  or  used  to  fortify 
sweet  wines.  The  principal  fruits  used  besides  grapes  are  apples, 
peaches,  plums  and  cherries. 

Industrial  alcohol  has  been  made  from  inferior  or  spoiled  fruits  and 
from  cannery  waste*,  but  the  cost  per  unit  of  alcohol  is  usually  high. 
The  difficulties  of  fermentation  are  great,  owing  to  the  presence  of  large 
quantities  of  molds  and  other  injurious  organisms,  and  the  extraction 
of  the  juice  is  troublesome.  A  careful  use  of  sulphites  and  pure  yeast 
much  simplifies  the  process. 

Sugar  cane  and  its  products  are  used  in  several  ways  to  produce  alcohol.  To  a 
limited  extent  the  juice  of  the  cane  is  fermented  directly  and  distilled.  The  product 
is  known  as  Jamaica  rum.  Much  larger  quantities  of  alcohol  are  manufactured  from 
the  cane-sugar  molasses  and  appear  in  commerce  as  rum,  tajfia,  arrack  or  neutral 
spirits. 

For  the  making  of  Jamaica  rum  the  juice  is  pressed  from  the  crushed  canes,  and 
diluted  with  20  per  cent  of  vinasses  (the  residue  of  a  previous  distillation)  to  increase 
the  acidity,  and  give  the  required  flavor. 

Cane  molasses  which  contain  from  50  to  60  per  cent  of  fermentable  sugar  are 
diluted  with  water  or  vinasses  to  i5°-i8°Bal.  and  partially  neutralized  with  lime 
when  the  acidity  is  excessive. 

One  of  the  principal  sources  of  industrial  alcohol  is  the  sugar  beet.  This  alcohol  is 
also  used  for  the  adulteration  or  imitation  of  potable  spirits. 

It  may  be  made  by  the  direct  fermentation  of  the  beet  juice,  extracted  by  grinding 
and  pressing,  by  methodical  maceration  or  by  diffusion.  Sulphuric  acid  is  added 
during  extraction.  This  facilitates  the  extraction  by  setting  free  organic  acids,  and 
represses  the  growth  of  injurious  microorganisms.  The  amount  used  should  be  such 
that  a  minute  quantity  of  sulphuric  acid  remains  free. 

Most  beet  alcohol  is  made  from  the  coarser  molasses  of  the  sugar  factories.  The 
molasses  are  diluted  to  2o°-3o°  Bal.  with  water,  further  diluted  and  heated  with 


MISCELLANEOUS  ALCOHOLIC  BEVERAGES  AND  PRODUCTS  633 

steam  and  acidified  with  sulphuric  acid.  The  sulphuric  acid  neutralizes  the  lime 
which  has  been  used  in  the  manufacture  of  the  sugar,  sets  free  the  volatile  acids  and 
breaks  up  the  nitrites  producing  nitrogen  peroxide.  The  liquid  is  then  boiled  for 
about  one  quarter  of  an  hour  to  drive  off  the  volatile  acids  and  the  oxides  of 
nitrogen  which  would  prevent  yeast  fermentation.  The  liquid  after  cooling  is  then 
fermented  with  yeast. 

Starchy  Raw  Materials. — In  the  preparation  of  a  fermentable  solu- 
tion from  starchy  materials  three  methods  for  the  conversion  of  the 
starch  into  sugar  may  be  used,  depending  respectively  on  the  action  of 
malt,  dilute  mineral  acids,  and  certain  molds. 

The  malt  used  in  saccharification  may  be  made,  in  a  manner  similar  to  that 
described  for  brewing,  from  barley,  oats,  rye  or  maize.  As  the  object  in  this  case  is  to 
cause  complete  conversion  of  the  starch  with  as  little  malt  as  possible,  the  malt 
should  have  the  maximum  diastatic  power.  For  this  reason,  germination  should  be 
carried  further  than  for  brewing  and  the  malt  used  green.  Drying  the  malt  de- 
stroys half  its  diastase. 

The  conversion  may  also  be  accomplished  by  boiling  one  part  of  grain  in  four 
parts  of  water  with  hydrochloric  or  sulphuric  acid.  With  the  former  acid,  10  per 
cent  of  the  weight  of  the  grain  is  used  and  5  per  cent  with  the  latter.  The  con- 
version requires  from  eight  to  twelve  hours'  boiling.  The  starch  is  first  converted 
into  dextrins  and  then  into  glucose.  If  the  boiling  is  too  prolonged  some  of  the  glucose 
may  be  lost  by  conversion  into  caramel.  The  amount  of  acid  and  the  time  of  boil- 
ing may  be  much  reduced  by  operating  under  2  to  3  kg.  pressure.  In  this  case  200 
liters  of  water  are  heated  with  100  kg.  of  grain  and  4  kg.  of  acid.  Conversion  occurs 
in  from  40  to  60  minutes.  The  acidity  is  reduced  with  calcium  oxide  or  calcium 
carbonate  before  fermentation. 

The  power  of  certain  molds,  especially  mucors,  to  convert  starch  into 
sugar  has  been  utilized.  Mucor  rouxii  found  in  Chinese  yeast,  Mucor 
oryza  in  Ragi,  and  related  forms  have  been  used  for  this  purpose.  This 
is  known  as  the  Amylo  Process.  The  grain  is  first  soaked  for  a  few 
hours,  then  heated  with  twice  its  weight  of  water  under  a  pressure  of 
three  and  a  half  to  four  atmospheres  until  soft  and  the  starch  rendered 
soluble.  The  liquefaction  of  the  starch  is  facilitated  by  slightly  acidu- 
lating the  water  with  hydrochloric  acid.  The  mixture  is  then  cooled 
to  38°  and  inoculated  with  a  pure  culture  of  the  Mucor.  A  current  of 
filtered  air  is  then  passed  through  the  mass  for  twenty-four  hours,  by 
which  time  the  mycelium  has  permeated  the  mass.  The  temperature 
is  then  reduced  to  33°,  pure  yeast  added  and  aeration  continued  for 
twenty-four  hours  longer  to  promote  the  multiplication  of  the  yeast. 
Conversion  of  the  starch  and  fermentation  of  the  sugar  then  continue 


634  MICROBIOLOGY   OF   ALCOHOLIC   FERMENTATION 

together.     The  mucor  is  capable  of  fermenting  the  sugar  and  producing 
alcohol,  but  the  yeast  acts  more  rapidly. 

The  malting  process  fe  the  most  commonly  employed.  The  acid  process  de- 
stroys a  greater  part  of  the  value  of  the  residues  of  distillation  and  the  amylo  process, 
requiring  costly  special  equipment  and  large  expenditures  for  fuel,  has  not  come 
into  genejral  use. 

The  starchy  substances  used  being  usually  neutral  or  of  low  acidity 
the  sugar  solutions  produced  would  be  very  liable  to  bacterial  invasion 
unless  means  of  prevention  were  used. 

In  the  amylo  process,  the  sterilization  of  the  solutions  and  the  use  of 
pure  cultures  accomplish  this  end.  In  the  acid  process,  the  minute 
quantity  of  free  mineral  acid  remaining  in  the  completed  solution  pre- 
vents any  considerable  growth  of  bacteria.  In  the  malting  process  the 
injurious  bacteria  are  restrained  by  lactic  acid  produced  by  lactic 
bacteria,  originating  in  the  malt  or  in  the  yeast  starter.  The  requisite, 
bacteria  are  obtained  by  keeping  the  starter  or  mother  yeast  at  50° 
to  58°  for  a  certain  time.  This  is  a  favorable  temperature  for  lactic 
and  too  high  for  the  development  of  acetic  or  other  injurious  bacteria. 
When  the  acidity  of  the  solution  reaches  3.5  g.  to  5  g.  per  liter  the  danger- 
ous butyric  bacteria  cannot  develop. 

Pure  lactic  acid  may  be  added  immediately  after  saccharification  and 
the  loss  of  sugar  due  to  the  action  of  the  lactic  bacteria  avoided,  but 
the  high  cost  of  the  pure  acid  prevents  the  practice. 

Yeast  being  much  less  sensitive  to  the  presence  of  certain  antiseptics 
than  bacteria  it  is  possible  to  control  the  latter  by  the  addition  of 
suitable  amounts  of  an  antiseptic  to  the  sugar  solution.  In  certain  cases 
moreover  by  gradually  increasing  the  amount,  yeast  can  be  accustomed 
to  concentrations  of  antiseptics  which  render  the  growth  of  bacteria 
impossible.  In  Effront's  method  for  the  preparation  of  distillation 
material,  hydrofluoric  acid  is  used.  This  acid  is  added  to  the  mother 
yeast  at  the  rate  of  10  g.  per  hectoliter  and  to  the  sugar  solution  in 
somewhat  smaller  amounts.  This  results  in  the  inhibition  of  lactic, 
butyric  and  other  bacteria  and  an  increase  in  the  fermentative  power  of 
the  trained  yeast. 

FERMENTATION. — The  sugar  solution  properly  diluted  and  acetified 
or  sterilized  is  fermented  by  the  addition  of  a  mother  yeast,  usually 
taken  from  a  previous  fermentation. 


MISCELLANEOUS    ALCOHOLIC    BEVERAGES    AND    PRODUCTS   635 

:The  original  yeast  may  be  obtained  by  a  spontaneous  fermentation 
as  is  usual  in  the  manufacture  of  rum.  Such  a  yeast  is  always  impure, 
containing  various  yeasts,  molds  and  bacteria,  and  is  therefore  very 
variable  and  uncertain  in  its  results. 

In  the  fermentation  of  beet  juice  and  beet  molasses,  beer  yeast  of  the 
Frohberg  type  or  special  distillers  yeasts  are  used.  A  starter  or  mother 
yeast  is  prepared  for  each  vat  or  the  process  is  made  continuous  by 
leaving  one-third  to  one-half  of  the  contents  of  a  fermented  vat  to  start 
a  fresh  addition  of  the  sugar  solution.  With  the  latter  method  the  yeast 
in  time  becomes  weak  and  badly  contaminated  and  a  new  start  must 
be  made  with  fresh  yeast. 

In  the  fermentation  of  solutions  made  from  potatoes,  corn  or  other 
starchy  substances,  each  vat  is  started  with  a  mother  yeast.  The  tem- 
perature should  be  kept  below  30°  by  means  of  refrigeration/otherwise 
alcohol  will  be  lost  by  the  multiplication  of  bacteria. 

By  the  use  of  pure  yeast,  the  yield  in  alcohol  is  greater  as  no  sugar  is 
wasted  in  the  production  of  lactic  acid.  The  cost,  however,  is  greater 
owing  to  the  necessity  of  the  use  of  more  heat  in  sterilization. 

The  fermentation  of  sugar-cane  molasses  for  the  production  of 
arrack  is  brought  about  by  the  use  of  a  mother  yeast  called  tapej, 
prepared  from  ragi  or  Java  yeast* 

Tapej  is  made  by  mixing  powdered  ragi  with  boiled  rice.  In  two 
days  the  rice  is  reduced  to  a  semi-fluid  condition  and  contains  bacteria, 
molds  and  yeasts.  The  bacteria  seem  to  have  no  part  in  the  process  but 
when  too  numerous  are  injurious.  The  mold  Mucor  oryza  converts  the 
rice  starch  into  sugar  and  the  yeast  S.  wrdemanni  produces  alcohol  from 
the  sugar.  The  other  molds  present  are  more  or  less  injurious. 


CHAPTER  IV 
THE  MANUFACTURE  OF  VINEGAR 

ACETIC  FERMENTATION 

NATURE  AND  ORIGIN  OF  VINEGAR. — Vinegar  is  a  condiment  made 
from  various  sugary  or  starchy  matters  by  alcoholic  and  subsequent 
acetic  fermentation.  It  should  contain  from  4  to  8  per  cent  of  acetic 
acid  and  natural  flavoring,  coloring  and  other  matters,  varying  accord- 
ing to  its  origin. 

Acetic  acid  (CH3COOH)  is  a  monobasic  organic  acid,  the  second  in 
the  fatty  acid  series.  It  is  a  colorless  liquid  with  a  strong  suffocating 
odor,  crystalizing  when  pure  at  16.7°  and  at  lower  temperatures  when 
diluted  with  from  i  to  13  per  cent  of  water.  Its  specific  gravity  is  1.08 
at  o°  and  it  boils  at  118°  under  760  mm.  pressure,  producing  an  inflam- 
mable vapor.  It  is  a  solvent  of  many  organic  substances  and  is  soluble 
in  water  and  alcohol  in  all  proportions. 

The  metallic  acetates  are  poisonous  and  are  formed  in  most  cases 
by  simple  contact  of  metal  and  acid.  Certain  alloys  of  tin  resist  the 
action  of  the  acid. 

Acetic  acid  is  formed  by  the  oxidation  of  ethyl  alcohol  which  takes 
place  in  two  stages  according  to  the  following  reactions: 

2C2H5OH  -f    02^  =  2CH3CHO  +  2H2O 

Ethyl      +  Oxygen  =       Acetic     -f  Water, 
alcohol  aldehyde 

2CH3CHO  +  O2     =  2CH3COOH 

Acetic     +  Oxygen  =  Acetic  acid, 
aldehyde 

These  reactions  may  be  brought  about  by  chemical  means,  but  in 
practice  they  are  due  to  the  action  of  certain  microorganisms;  mainly 
bacteria.  Acetic  acid  is  also  made  by  the  distillation  of  wood  but  the 
product  is  not  suitable  for  consumption. 

VINEGAR  BACTERIA. — If  wine,  beer  or  a  similar  organic  solution  con- 
taining alcohol,  is  exposed  freely  to  the  air  it  soon  becomes  covered 
with  a  film3  the  alcohol  disappears,  is  replaced  by  acetic  acid  and  the 
liquid  is  converted  into  vinegar. 

636 


THE   MANUFACTURE    OF  VINEGAR  637 

This  film,  the  My  coder  ma  aceti  of  Pasteur,  consists  of  bacteria 
cohering  by  means  of  a  glutinous  sheath  surrounding  each  cell,  forming 
a  zooglea.  If  the  film  is  undisturbed,  the  liquid  remains  clear  until 
converted  into  vinegar,  if  disturbed,  portions  may  sink,  new  films  form 
and  finally  a  large  gelatinous  zoogleic  mass,  "the  mother  of  vinegar," 
may  form  in  the  liquid. 

Sometimes,  especially  on  liquids  containing  sugar  and  large  amounts 
of  alcohol,  such  as  sweet  wines,  the  film  formed  consists,  not  of  bacteria, 
but  of  a  yeast-like  fungus,  Mycoderma  mni. 

Wines  which  have  been  sterilized,  often  remain  without  acetifying 
for  a  considerable  time.  Those  containing  free  sulphurous  acid  acetify 
slowly  and  with  difficulty.  Ordinarily  at  warm  temperatures,  exposed 
wines  develop  a  bacterial  film  very  rapidly  owing  to  the  almost 
constant  presence  of  some  acetic  bacteria  in  all  wines. 

Hansen  was  the  first  to  show  that  the  vinegar  bacteria  included  more 
than  one  species.  He  isolated  and  described  three  species  concerned 
with  the  spontaneous  souring  of  beers.  Later  it  was  shown  by  A.  J. 
Brown,  Henneberg,  and  others  that  several  other  species  commonly 
occurred  in  vinegar  factories  and  that  many  more  were  capable  of  pro- 
ducing acetic  acid  in  small  amounts.  The  species  which  have  been 
most  thoroughly  studied  and  which  seem  to  occur  most  usually  in 
vinegar  factories  are  Bad.  aceti,  Bact.  pasteurianum,  Bact.  kutzingianum, 
Bact.  xylinum,  short  descriptions  of  which  follow: 

Bacterium  aceti  (Kiitzing),  Hansen.  This  species  consists  of  rods  about  in  or 
2/t  in  length,  somewhat  constricted  in  the  middle  and  lying  in  parallel  chains  in 
the  surface  film.  This  film  is  moist,  smooth,  veined,  and  forms  in  about  twenty- 
four  hours  at  34°.  On  wort  gelatin,  it  forms  gray,  waxy,  raised  colonies  which  are 
usually  round,  with  unbroken  edges  but  sometimes  star-shaped  and  consisting  of 
separate  rod-shaped  cells. 

Bacterium  pasteurianum,  Hansen. — The  cells  of  this  species  are  somewhat  larger 
than  those  of  aceti  and  more  commonly  produce  thread-like  and  swollen  involution 
forms.  The  film  is  dry  and  soon  becomes  wrinkled.  Colonies  on  wort  gelatin  are 
smaller  than  those  of  aceti,  rugose,  and  the  cells  retain  their  arrangement  in  chains. 
The  mucilaginous  sheath  is  stained  blue  with  iodine-potassium  iodide  solution 
(saturated  solution  of  KI  colored  brown  by  the  additionof  a  few  drops  of  an  alcoholic 
solution  of  I),  in  this  differing  from  Bad.  aceti  (Fig.  158). 

Bacterium  kutzingianum,  Hansen. — The  cells  resemble  those  of  Bact.  aceti,  but 
are  usually  free  or  in  pairs.  The  film  resembles  that  of  Bact.  aceti  but  has  a  tend- 
ency to  climb  up  the  sides  of  the  flask  above  the  liquid.  The  colonies  on  wort  gelatin 
are  smooth  and  shiny.  The  mucilage  stains  blue  with  the  iodine  solution. 


638 


MICROBIOLOGY   OF  ALCOHOLIC   FERMENTATION 


Bacterium  xylinum,  A.  J.  Brown. — This  species  forms  a  thick  tough,  leathery  film, 
the  gelatinous  substance  of  which  stains  blue  with  iodine  and  sulphuric  acid. 
B.  acetigenus,  B.  oxydans,  and  B.  industrius  are  motile  species. 

All  species  are  strictly  aerobic  and  grow  quickly  only  when  freely 
supplied  with  oxygen.  This  oxygen  is  necessary  for  the  acetification 
of  the  alcohol.  Duclaux  has  calculated  that  one  centigram  of  the 
bacterial  film  is  capable  of  uniting  1.3  g.  of  oxygen  to  alcohol,  130  times 
its  own  weight.  The  optimum  temperature  for  most  species  is  about 


FIG.  158. — Vinegar  bacteria.  A,  Bact.  aceti;  B,  Bact.  pasteurianum;  C/  Bad. 
kiitzingianum;  D,  Bact.  pasteurianum,  showing  mucilaginous  sheath.  (After 
Hansen.) 

34°  and  the  range  of  temperature  at  which  they  grow  is  between  4° 
and  7°  to  42°.  They  all  form  acetic  acid  from  ethyl  alcohol,  propionic 
acid  from  propyl  alcohol  and  most  of  them  gluconic  acid  from  dextrose. 
B.  industrius  and  B.  oxydans,  according  to  Henneberg,  can  form  acids 
from  a  large  number  of  sugars  and  related  substances,  including 
saccharose,  maltose,  starch,  dextrin,  glycerin  and  mannit. 

The  presence  of  too  much  alcohol  prevents  the  growth  of  acetic 
bacteria,  the  limit  being  about  14  per  cent  under  manufacturing  con- 
ditions. At  14  per  cent  and  above,  the  film  forms  with  difficulty,  and 
the  oxidation  of  the  alcohol  is  incomplete,  aldehyde  and  irritating 
products  being  formed.  Acetic  acid  in  amounts  above  10  to  12  per 
cent  is,  moreover,  antiseptic  to  the  bacteria.  Below  14  per  cent  of 
alcohol,  the  bacteria  develop  readily  and  produce  in  suitable  solutions, 
besides  acetic  acid,  agreeable  ethers  which  are  more  abundant  when 
the  oxidation  is  slow.  Below  i  or  2  per  cent  of  alcohol,  the  bacteria 


THE   MANUFACTURE    OF  VINEGAR  639 

attack  these  ethers,  and  finally  the  acetic  acid  itself,  causing  complete 
oxidation  according  to  the  equation: 

CH3COOH  +  40  =  2CO2  -f  2H2O. 

The  addition  of  a  new  supply  of  alcohol,  however,  immediately 
arrests  this  reaction.  In  practice  the  acetification  should  be  stopped 
when  the  alcohol  has  fallen  to  i  or  2  per  cent,  otherwise  there  is  a  loss 
of  flavor  and  of  acetic  acid,  which  may  continue  until  all  the  acid  is 
destroyed. 

The  length  of  time  during  which  the  acetic  bacteria  retain  their 
vitality  varies  with  the  moisture  and  the  temperature.  In  nutrient 
solutions,  they  live  from  one  to  as  many  as  ten  years;  in  the  dry  state, 
from  three  months  at  ordinary  temperatures,  to  twelve  months  at  2°. 

PROCESSES  OF  MANUFACTURE 

RAW  MATERIALS. — Originally  vinegar  was  made  from  wine,  as 
indicated  by  the  etymology  of  the  word  which  means  " acetified  wine." 
Later,  other  alcoholic  beverages  such  as  cider  and  beer  were  used  for 
the  same  purpose.  In  these  liquids,  the  acetic  bacteria  find  all  the 
mineral  and  organic  matters  necessary  for  their  development,  together 
with  alcohol  in  amounts  favorable  for  acetic  fermentation.  At  present, 
a  large  number  of  materials  containing  alcohol,  or  starchy  and  sugary 
matters,  which,  by  preliminary  yeast  fermentation,  can  be  changed 
into  alcohol  are  used  as  sources  of  vinegar.  The  most  important  of 
these  are  honey,  malt,  and  various  fruit  juices. 

All  these  materials  make  wholesome  vinegar  of  varying  degrees  of 
quality.  Those  of  wine  and  cider  are  usually  classed  as  the  best,  and 
those  of  malt  and  honey  next.  The  great  bulk  of  the  vinegar  of  com- 
merce, however,  at  present  is  made  by  the  acetification  of  distilled 
grain,  potato  and  molasses  alcohol.  This  is  not  vinegar  strictly  speak- 
ing but  an  imitation,  consisting  of  a  dilute  solution  of  acetic  acid  with- 
out the  various  flavors  which  are  an  essential  part  of  pure  vinegar.  In 
order  to  give  it  a  semblance  of  the  latter,  it  is  often  colored  with 
caramel  and  flavored  with  various  substances. 

Other  imitations  of  vinegar  sometimes  appear  on  the  market,  con- 
taining wood  vinegar,  or  even  mineral  acids.  These,  however,  are  more 
or  less  poisonous  and  their  sale,  as  food,  is  usually  forbidden  by  law. 


640  MICROBIOLOGY   OF   ALCOHOLIC   FERMENTATION 

FERMENTATION. — If  the  raw  material  to  be  used  is  starchy  or 
sugary,  it  must  be  first  changed  into  an  alcoholic  liquid  containing  from 
6  to  12  per  cent  of  alcohol  by  volume.  This  is  accomplished  by  one 
of  the  methods  discussed  in  the  preceding  chapter.  This  alcoholic 
fermentation  must  be  kept  rigidly  distinct  from  the  acetification  and  is 
best  carried  out  in  a  separate  building.  The  yeast  must  finish  its  work 
before  the  bacteria  commence  theirs.  The  reason  for  this  is  that  yeasts 
are  very  sensitive  to  acetic  acid  and  a  small  quantity  may  paralyze 
their  activity  and  prevent  the  change  of  all  the  sugar  into  alcohol, 
with  a  consequent  loss  of  strength  and  quality  in  the  final  product. 

The  quality  of  the  vinegar  will  depend  on  the  quality  of  the  raw 
material  from  which  it  is  made.  Wine  or  cider  spoiled  by  bacterial 
fermentation,  moldy  casks,  etc.,  will  make  inferior  vinegar.  An 
exception  to  this  may  be  made  of  so-called  "pricked"  wines,  which  are 
simply  wines  in  which  acetic  fermentation  has  started  spontaneously. 
The  wine  or  other  alcoholic  liquid  should  be  perfectly  clear  and  clean 
tasting  and,  if  necessary,  should  be  fined,  filtered  or  pasteurized  im- 
mediately before  use.  It  should  contain  no  antiseptic  which  would 
interfere  with  the  development  of  the  acetic  bacteria.  Sulphurous 
acid,  if  present  in  the  free  state,  should  be  removed  or  oxidized  by 
thorough  aeration. 

Commerical  alcohols  made  from  corn,  potatoes,  beets,  molasses  and 
other  products  can  be  used.  The  special  flavors  of  these  alcohols,  due 
to  their  origin,  disappear  almost  completely  in  the  vinegar.  This, 
however,  is  not  true  of  denatured  alcohol  or  that  containing  methyl 
alcohol  or  acetone. 

The  alcohol  must  be  diluted  to  from  10  to  1 2  per  cent  by  volume,  and 
then  made  suitable  for  the  growth  of  acetic  bacteria  by  the  addition  of 
nutritive  substances  containing  nitrogen  and  phosphates.  This  is  ac- 
complished usually  by  adding  10  per  cent  of  wine,  beer,  malt-extract, 
yeast  decoction,  or  similar  material  to  the  diluted  alcohol.  The  waste 
liquids  from  a  brandy  distillery  may  be  used  instead  of  water  for  dilution. 
After  resting  a  few  days,  the  mixture  is  filtered  and  is  then  ready  for 
acetification. 

Before  starting  the  acetic  fermentation,  it  is  a  usual  and  good 
practice  to  add  about  10  per  cent  of  good  vinegar  to  the  liquid,  which  is 
thus  rendered  acid  and  therefore  less  liable  to  alteration  by  injurious 
bacteria  and  other  microorganisms. 


THF    MANUFACTURE   OF  VINEGAR  641 

All  the  processes  of  vinegar-making  depend  on  the  same  principle, 
which  is  to  expose  the  liquids  prepared  as  above  to  the  action  of  acetic 
bacteria  with  full  access  of  atmospheric  oxygen  at  a  suitable  tem- 
perature. The  rapidity  of  the  process  depends  on  the  number  of 
active  bacteria  present,  the  nutritive  value  of  the  liquid,  the  tem- 
perature, and  especially  on  the  free  access  of  oxygen. 

STARTERS  AND  PURE  CULTURES. — The  10  per  cent  of  vinegar  added 
to  the  liquid  to  be  fermented  usually  contains  sufficient  bacteria  to  in- 
sure a  prompt  start.  Where  this  is  not  the  case,  a  starter  may  be 
prepared  by  exposing  a  suitable  liquid  in  a  shallow  vessel  to  the  air  of  a 
warm  room  for  several  days.  Any  liquid  containing  about  4  per 
cent  of  alcohol,  2  per  cent  of  acetic  acid  and  a  moderate  amount  of 
nitrogenous  matter  is  suitable.  A  decoction  made  by  boiling  50  g. 
of  fresh  yeast  in  1,000  c.c.  of  water,  filtering  and  adding  the  proper 
amount  of  vinegar  and  wine  or  beer  will  serve.  After  thorough 
aeration,  such  a  liquid  in  a  few  days  becomes  covered  with  a  film  of 
acetic  bacteria.  This  film  may  be  used  as  a  starter  by  gently  submerg- 
ing the  vessel  in  which  it  is  formed  in  the  liquid  to  be  acetified,  or  by 
removing  with  a  clean  sliver  of  wood  which  is  afterward  floated  in  the 
liquid. 

In  practice,  such  a  starter  gives  a  sufficiently  pure  fermentation  of 
acetic  bacteria.  The  particular  species  of  acetic  bacteria,  however,  is 
left  to  chance.  Pure  cultures  of  a  particular  selected  form  would  in  all 
probability  improve  the  certainty  of  the  production  of  good  vinegar, 
but  the  method  has  not  entered  into  general  practice. 

APPARATUS. — Most  metals  of  all  kinds  should  be  avoided  as  much 
as  possible.  The  hoops  of  barrels  and  buckets  may  be  protected  by  a 
coating  of  paraffin.  Pumps  may  be  of  wood  or  of  the  special  alloys  al- 
ready mentioned,  or  they  may  be  so  constructed  that  they  will  not  come 
in  contact  with  the  liquids. 

METHODS 

DOMESTIC  METHOD. — A  cask  of  convenient  size  (40  to  200  liters)  is 
fitted  as  illustrated  in  Fig.  159. 

The  wine  or  cider  to  be  acetified,  after  filtering,  if  necessary,  is 
poured  into  the  cask  until  it  is  about  one-half  to  two-thirds  full,  the 
object  being  to  have  as  large  a  surface  as  possible  for  the  growth  of  the 
bacterial  film.  Free  circulation  of  air  is  insured  by  a  5-cm.  hole  in 
each  head  of  the  cask,  one  near  the  surface  of  the  liquid  and  one  near 

41 


642 


MICROBIOLOGY   OF  ALCOHOLIC   FERMENTATION 


the  top  of  the  cask.     These  holes  should  be  covered  with  varnished 
metal  netting  to  prevent  the  entrance  of  flies. 

The  top  bung  hole  is  then  closed  with  a  cork,  through  which  a  funnel 
passes,  furnished  at  its  lower  end  with  a  glass  tube  extending  to  within 
a  few  inches  of  the  bottom  of  the  cask.  By  means  of  this  funnel 
new  liquid  can  be  added  without  disturbing  the  surface  film.  The 
lower  bung-hole  is  closed  with  a  cork,  through  which  passes  an 
L-shaped  glass  tube  which  serves  as  an  indicator  of  level  and  which 
also  can  be  used  to  draw  off  the  vinegar. 


FIG.  159. — Vinegar  barrel.  Z,,  Surface  of  liquid;  0,  O,  openings  for  entrance  of 
air;  t,  tube  for  introducing  new  supplies  of  wine  without  disturbing  surface  films; 
E,  glass  tube  to  show  level  of  liquid  and  for  drawing  off  vinegar.  (Original.) 


When  this  apparatus  is  working  well,  from  one-fifth  to  one-quarter 
of  the  contents  may  be  taken  off  every  three  or  four  weeks.  This 
depends  on  the  temperature,  which  should  be  between  10°  and  18°. 
The  vinegar  drawn  off  is  immediately  replaced  with  wine  or  cider  which, 
if  added  slowly,  will,  owing  to  its  lower  specific  gravity,  remain  at  the 
surface  in  contact  with  the  bacterial  film. 

ORLEANS  METHOD. — This  is  practically  the  same  as  the  method  just 
described  with  slight  modifications  to  adapt  it  to  large  scale  opera- 
tions. It  is  the  oldest  commercial  method  and  produces  vinegar  of  the 
highest  quality. 

Barrels   of  about   two   hectoliters   are   usually   employed,   fitted 


THE   MANUFACTURE    OF  VINEGAR  643 

essentially  like  that  already  described  but  with  the  omisson  of  the 
funnel  and  drawing-off  tubes. 

The  wine  is  first  cleared  in  a  vinegar  filter.  This  consists  of  a 
wooden  vat  filled  with  beech  chips  which  have  been  extracted  by  soak- 
ing for  several  days  in  cold  water.  The  wine  remaining  in  contact 
with  these  chips  for  three  or  four  days  deposits  most  of  its  sediment. 

The  cask  is  first  one-third  filled  with  good  vinegar  and  10  or  15 
1.  of  the  filtered  wine  added.  The  same  amount  of  wine  is  added  every 
week  for  four  weeks  by  which  time  the  cask  is  half  full.  At  the  end  of 
the  fifth  week  an  amount  of  vinegar  equal  to  the  wine  added  is  drawn 
off  and  the  operation  repeated.  The  vinegar  is  filtered  as  soon  as  it  is 
drawn  off,  placed  in  full  tightly  bunged  casks  and  kept  in  a  cool 
cellar. 

PASTEUR  METHOD. — Pasteur  long  ago  pointed  out  the  defects  of  the 
old  Orleans  method  and  suggested  improvements.  The  main  defects 
of  the  old  method  are  that  it  is  cumbersome,  laborious,  slow  and  costly. 
There  is  a  loss  of  about  10  per  cent  of  material  by  evaporation  and  the 
repeated  additions  of  liquid  break  the  bacterial  film,  which  then  sinks 
to  the  bottom,  grows  anaerobically  and  exhausts  the  nutrients  of  the 
solution  without  producing  acetic  acid.  These  submerged  bacteria 
finally  form  a  large  gelatinous  mass  which  interferes  with  the  regular 
progress  of  the  operations,  depreciates  the  quality  and  necessitates 
frequent  expensive  cleanings  of  the  casks.  Many  attempts,  more  or  less 
successful,  to  overcome  these  defects  in  accordance  with  Pasteur's  ideas 
have  been  made,  that  of  Claudon  is  one  of  the  best  and  will  serve  to 
exemplify  all. 

It  consists  essentially  of  a  wide,  shallow,  covered  square  vat, 
furnished  with  numerous  openings  near  the  top  by  which  the  entrance 
of  air  can  be  facilitated  and  regulated..  This  vat  is  filled  to  the  bottom 
of  the  air  vents  with  a  mixture  of  four  parts  of  good  new  vinegar  and 
six  parts  of  wine  which  has  been  pasteurized  at  55°  and,  when  necessary, 
filtered.  On  top  of  this  liquid  is  floated  a  light  wooden  grating  which 
helps  to  support  the  bacterial  film  and  prevent  its  breaking  and  sub- 
merging during  the  various  operations.  When  filled,  the  process  is 
started  by  placing  a  small  quantity  of  a  good  bacterial  film  on  top  of 
the  liquid  which  soon  becomes  completely  covered  when  the  proper 
conditions  of  temperature  and  aeration  are  maintained. 

Each  acetifying  vat  is  connected  with  a  small  measuring  vat  from 


644  MICROBIOLOGY   OF   ALCOHOLIC   FERMENTATION 

which  the  proper  amount  of  liquid  is  added  every  day  after  a  correspond- 
ing amount  of  vinegar  has  been  removed.  These  two  vats  constitute  a 
unit,  several  of  which,  usually  six,  are  united  in  a  battery.  A  factory 
includes  several  of  these  batteries. 

The  batteries  are  fed  from  a  large  vat  or  reservoir,  where  the  mixture 
of  wine  and  vinegar  is  prepared  and  stored.  The  vinegar  drawn  from 
the  batteries  runs  directly  to  filters,  thence  to  a  pasteurizer,  and  finally 
to  the  storage  casks. 

The  output  of  these  batteries  is  from  two  to  five  times  as  great  per 
square  meter  of  acetifying  surface  as  that  of  the  old  method;  the  cost 
of  the  operation  is  considerably  less,  the  loss  by  evaporation  much 
reduced  and  the  quality  equal  and  much  more  under  control  of  the 
manufacturer. 

RAPID  METHODS. — In  all  the  methods  described,  the  surface  of  the 
liquid  exposed  to  air,  where  alone  acetification  occurs,  is  small  compared 
to  the  volume  of  the  liquid.  In  order  to  hasten  and  therefore  cheapen 
the  process,  various  devices  for  increasing  the  surface  in  contact  with 
air  have  been  devised.  The  simplest  of  these  is  one  sometimes  em- 
ployed in  wine-making  countries.  The  pressed  pomace  of  red  wine  is 
broken  up  and  placed  loosely  but  uniformly  in  a  tall  narrow  vat.  In  a 
few  days,  acetic  fermentation  commences  in  all  parts  of  the  mass. 
Wine  is  then  sprinkled  periodically  on  top  and  trickling  down  over  the 
pomace,  it  is  changed  to  vinegar  by  the  bacterial  film  which  encases 
every  particle  of  the  mass.  The  "  quick  "  or  German  method  of  vinegar- 
making  is  based  on  this  principle. 

The  apparatus  used  in  this  method  consists  of  a  tall  cylindrical  or 
slightly  conical  wooden  vat  provided  with  a  perforated  false  head  a  few 
inches  from  the  bottom  and  another,  similar  in  structure,  at  the  same 
distance  from  the  top.  The  space  between  these  two  false  heads  is  filled 
with  long  thin  chips  or  shavings  of  beech  wood  which  have  been  thor- 
oughly extracted,  first  with  water  and  then  with  good  strong  vinegar 
(Fig.  1 60).  Various  substitutes  for  beech  chips  have  been  used  with 
more  or  less  success.  Rattan  shavings  and  wastes  are  suitable;  dried 
corn-cobs  can  be  used  but  are  not  durable;  wood  charcoal  in  lumps  is 
used  successfully;  coke  is  as  effective  as  wood  charcoal  and  more 
durable  and  is  now  used  extensively  in  the  manufacture  of  alcohol 
vinegar. 

In  operation,  the  liquid  to  be  acetified  is  distributed  over  the  top 
false  head  intermittently  in  small  amounts.  This  intermittence  of 


THE    MANUFACTURE    OF  VINEGAR 


645 


c 


anana 


qp 


•  •"• Vid  o 


FIG.  1 60. — Rapid  process  vinegar  apparatus.  V,  Mass  of  beech  chips  over  which 
the  alcoholic  liquids  run  from  H;  H,  false  head  with  numerous  small  holes  and  threads 
for  the  slow  and  equal  distribution  of  the  liquid;  E,  tilting  trough  for  the  intermittent 
supply  of  liquid;  o0  openings  for  the  entrance  and  exit  of  air.  |  Path  °f  air5 
-*  path  of  liquid.  (Original.) 


646  MICROBIOLOGY   OF   ALCOHOLIC   FERMENTATION 

supply  is  accomplished  by  various  automatic  devices.  If  the  supply  is 
continuous,  the  liquid  tends  to  run  in  streams  or  currents  in  certain 
parts  of  the  vat  and  much  of  the  acetifying  surface  is  lost;  if  too  rapid, 
the  bacterial  film  is  removed  from  the  upper  part  of  the  mass  of  beech 
chips  and  only  the  lower  part  is  effective. 

From  the  false  head,  the  liquid  passes  through  numerous  small  holes 
to  the  mass  of  beech  chips,  over  which  it  trickles  slowly  and  is  acetified 
by  means  of  the  bacterial  film  which  covers  them.  By  the  time  it 
reaches  the  lower  false  head,  the  alcohol  is  in  greater  or  less  amount 
converted  into  acetic  acid.  Usually  the  liquid  must  pass  through  from 
two  to  five  times  or  through  an  equal  number  of  vats  before  it  is  com- 
pletely changed  into  vinegar.  The  number  of  passages  depends  on  the 
amount  of  alcohol  present,  the  height  of  the  acetifying  column,  the 
rapidity  of  the  flow,  the  temperature,  and  on  the  perfection  of  the 
apparatus. 

Oxygen  is  supplied  by  the  air  which,  entering  holes  in  the  vat  below 
the  lower  false  head,  passes  through  numerous  holes  in  the  latter, 
through  the  interstices  between  the  chips  and  out  through  short  tubes 
fixed  in  the  upper  false  head  and  holes  in  the  top.  The  passage  of  air 
is  insured  by  the  heating  of  the  interior  due  to  the  fermentation.  It 
can  be  regulated  by  the  number  and  diameter  of  the  air  holes. 

The  temperature,  which  should  be  close  to  30°,  must  be  carefully 
regulated.  If  the  temperature  rises  too  high,  the  loss  by  evaporation 
will  be  much  increased;  if  it  remains  too  low  the  acetification  will  be 
retarded.  Too  low  a  temperature  is  less  injurious  than  too  high  a 
temperature. 

Many  modifications  of  this  method  exist,  having  principally  for 
their  objects  the  more  complete  regulation  of  the  temperature  and  air 
supply,  the  recuperation  of  the  volatile  matters,  and  the  avoidance  of 
the  need  of  repassing  the  liquid  through  different  acetifying  columns. 

ROTATING  BARRELS. — Several  methods  are  in  use  which  attempt  to 
combine  the  rapidity  of  the  German  machines  with  the  quality  of  the 
Orleans  method  and  which  are  suitable  for  use  with  wine  and  cider. 
These  liquids  cannot  be  acetified  conveniently  by  the  German  method 
on  account  of  the  large  amounts  of  solids  and  extractive  matter  they 
contain.  These  coat  the  beech  chips  rapidly  and  interferes  with  the 
perfect  working  of  the  machine. 

These  methods  make  use  of  a  barrel  filled  partially  or  wholly  with 


THE   MANUFACTURE    OF  VINEGAR  647 

beech  chips  and  half  filled  with  the  liquid  to  be  acetified.  By  rotating 
the  barrel  at  short  intervals  the  liquid  trickles  repeatedly  over  the  chips 
and,  with  proper  aeration,  the  acetification  is  rapid  and  complete.  The 
same  principle  has  been  applied  successfully  by  means  of  "drums"  10 
feet  long  by  4  feet  in  diameter  partially  immersed  and  rotating  in 
the  liquid  of  a  closed  vat  to  which  air  is  admitted  by  adjustable  holes. 

FUNCTION  or  THE  FILM. — All  these  methods  are  based  on  the  suppo- 
sition that  the  formation  of  acetic  acid  depends  on  the  work  of  the  bac- 
terial film  at  the  surface  of  the  alcoholic  liquid.  It  seems  probable 
that  the  only  function  of  the  film  is  to  maintain  the  bacteria  in  a  posi- 
tion where  they  can  obtain  a  full  supply  of  oxygen.  If  this  is  true, 
oxygen  supplied  by  a  stream  of  compressed  air  or  other  efficient  means 
of  aeration  should  be  equally  effective  even  in  the  absence  of  a  film. 

An  observation  by  W.  V.  Cruess  indicates  that  the  film  formation 
may  actually  hamper  the  work  of  the  bacteria.  This  observation  is 
that  certain  forms  of  vinegar  bacteria  which  do  not  form  films  produce 
acetic  acid  very  rapidly.  The  observation  was  made  on  wine  in  small 
flasks  and  at  temperatures  of  2o°-33°.  Whether  the  formation  of 
acetic  acid  would  be  equally  rapid  in  larger  volumes  of  liquid  where  the 
penetration  of  oxygen  would  be  slower  has  not  been  determined. 

AFTER-TREATMENT. — Alcohol  vinegars  require  little  treatment. 
They  should  be  filtered  and  are  usually  colored  slightly  with  caramel. 
Being  little  more  than  dilute  solutions  of  acetic  acid  without  ethers  or 
bouquet,  there  is  no  object  in  aging  them. 

Wine  and  cider  vinegars,  for  the  best  results,  require  aging  and 
careful  treatment.  They  should  be  filtered  and  pasteurized  as  soon  as 
made  and  stored  in  clean  casks  which  are  well  bunged  and  kept  con- 
stantly full  in  a  cool  place  of  even  temperature.  If  too  dark  in  color, 
they  may  be  decolorized  with  pure  animal  charcoal  carefully  extracted 
with  acids  and  water. 

Before  using  or  bottling,  the  vinegar  should  be  fined  with  isinglass 
(see  page  620). 

DISEASES 

The  most  troublesome  pest  of  vinegar  factories  is  a  minute  nema- 
tode,  the  Anguillula  aceti  or  vinegar  eel.  It  often  develops  around  the 
edges  of  the  surface  of  the  liquid  in  vinegar  barrels  and  in  the  acetify- 
ing columns  and,  if  neglected,  may  cause  putrefaction  and  spoiling  of 


648  MICROBIOLOGY    OF   ALCOHOLIC    FERMENTATION 

the  vinegar.  Frequent  and  thorough  cleaning  of  all  apparatus,  pasteur- 
ization of  liquids  and  light  sulphuring  of  empty  casks  will  prevent  its 
development.  The  vinegar  eels  are  easily  killed  by  heating  the  vinegar 
to  50°.  They  may  be  removed  by  nitration  or  fining. 

Microscopic  mites  are  sometimes  troublesome  in  neglected  factories. 
They  can  be  reduced  by  the  methods  recommended  for  vinegar  eels 
and  their  entrance  into  the  barrels  or  acetifying  columns  prevented  by 
painting  a  ring  of  turpentine  or  some  viscid  substance  around  each  air 
hole. 

Vinegar  flies  (Drosophylla  cellaris)  are  often  troublesome,  but  can  be 
excluded  by  proper  screening  of  buildings  and  barrels. 

Bacteria  other  than  acetic  may  develop  in  vinegar  and  some  of 
them  may  depreciate  its  quality.  These  have  been  little  studied  but 
the  most  harmful  seem  to  be  anaerobic  forms  which  develop  in  the 
lower  parts  of  the  liquid  protected  from  oxygen  by  the  screening  film 
of  the  acetic  bacteria.  They  produce  butyric  acid  and  putrid  odors 
and,  if  neglected,  may  completely  spoil  the  vinegar.  Sulphuring,  fining, 
and  pasteurization  are  the  remedies. 

Darkening  or  persistent  cloudiness  may  be  caused  by  oxidase  as  in 
wine  and  cider  and  is  controlled  in  the  same  way.  A  similar  defect  may 
be  caused  by  the  tannic  extractive  matters  of  new  casks  or  contact  with 
iron.  Aeration  followed  by  fining  will  remove  the  cause  of  the  trouble. 


DIVISION  VII 
MICROBIOLOGY  OF  SPECIAL  INDUSTRIES 


CHAPTER  I 

SPECIAL  INDUSTRIAL  FERMENTED  PRODUCTS 
ACETONE  AND  ACETIC  ACID* 

Prior  to  1914,  most  of  the  world's  supply  of  acetone  and  acetic 
acid  was  furnished  by  the  destructive  distillation  of  wood.  When 
wood  is  distilled  from  a  retort  at  high  temperatures  it  undergoes  a 
decomposition  by  which  acetone,  acetic  acid,  methyl  acetate,  and  other 
compounds  are  liberated  leaving  in  the  retort  crude  charcoal.  The 
acetic  acid  is  converted  into  calcium  acetate  by  neutralization  with 
lime.  The  acetic  acid  may  be  liberated  by  means  of  inorganic  acids 
and  distilled  to  give  acetic  acid  or  may  be  dried  and  destructively 
distilled  to  form  acetone.  Acetone  is  used  extensively  in  explosives 
manufacture  and  as  a  solvent  for  aeroplane  ''dope"  and  many  other 
organic  compounds.  It  is  useful  also  in  the  mixing  of  guncotton  and 
nitroglycerine  to  form  cordite,  the  explosive  used  so  extensively  by  the 
British  and  other  navies. 

Two  methods  of  producing  acetone  besides  wood  distillation  came 
into  common  use  during  the  war.  One  was  the  formation  of  acetone 
from  starchy  materials  by  direct  fermentation,  the  Fernbach  process; 
the  other  was  by  the  formation  of  acetic  acid  by  direct  bacterial  fer- 
mentation of  sea  kelp  or  the  alcoholic  and  acetic  fermentation  of  sugary 
material,  followed  by  conversion  of  the  acetic  acid  into  acetone. 

The  United  States  Industrial  (Jhemical  Company  at  Curtis  Bay 
during  the  war  produced  as  much  as  70,000  pounds  of  acetic  acid  per 
day  from  molasses  imported  by  tank  steamers  from  Cuba.  Over  1000 
beechwood  shaving-filled  generators  from  10X181018X25  feet  in 
size  were  necessary.  About  200,000  gallons  of  calcium  acetate  liquors 

*  Prepared  by  W.  V.  Cruess. 

649 


650  MICROBIOLOGY   OF   SPECIAL   INDUSTRIES 

per  day  were  concentrated  in  special  stills,  making  this  plant  the  largest 
of  its  kind  in  the  world.* 

In  the  Fernbach  process  corn  or  other  grains  are  subjected  to  a 
carefully  controlled  fermentation  process  in  which  acetone  is  formed 
directly.  It  is  not  clear  whether  the  acetone  is  formed  from  the  starch 
or  from  the  grain  proteins — probably  the  latter  figure  to  an  important 
degree.  Butyl  alcohol  is  formed  with  the  acetone.  It  is  stated  that 
100  tons  of  acetone  per  month  were  produced  during  the  war  in  a  plant 
in  Toronto  and  250  tons  per  month  in  the  United  States  by  this  process. 

Bacillus  macerans  is  used  in  a  process,  described  in  German  patent 
294683  (1914),  to  produce  acetone  from  wort.  Yeast  is  then  grown 
in  the  liquid  resulting  in  a  yield  of  both  yeast  and  acetone. 

Mezzadrolif  of  the  Sugar  Beet  Station  at  Rovigo,  Italy,  describes 
two  organisms,  B.  invertenti  lattici,  and  B.  invertenti  acetici,  which  are 
capable  of  forming  lactic  acid,  alcohol  and  acetone  directly  from  cane 
sugar.  Their  industrial  use  for  the  production  of  these  acids  from 
sugar  waste  is  advocated. 

The  Hercules  Powder  Company  and  other  companies  demonstrated 
that  acetone  and  acetic  acid  could  be  produced,  at  least  under  war-time 
conditions,  from  kelp  by  a  fermentation  method,  first  systematically 
investigated  by  L.  Lieb  and  D.  R.  Hoagland  of  the  University  of  Cali- 
fornia. The  giant  kelp  of  the  Pacific  ocean  was  harvested  by  a  "mow- 
ing machine  "-like  device,  taken  by  barges  to  the  plant  at  San  Diego, 
and  shredded  into  a  slimy  mass  of  pulp  and  juice.  This  mixture  was 
pumped  into  enormous  tanks  in  which  a  vigorous  growth  of  bacteria 
of  several  types  developed  spontaneously.  The  leaves  of  the  plant 
and  parts  of  the  stems  became  liquefied.  The  resulting  liquid  con- 
taining acetic  acid,  ethyl  acetate,  ethyl  propionate,  ethyl  butyrate, 
and  other  compounds  formed  during  fermentation  and  inorganic  salts 
was  neutralized  with  calcium  carbonate  forming  calcium  acetate.  This 
was  treated  with  sodium  sulphate  to  form  sodium  acetate  which  on 
concentration  was  crystallized  and  converted  into  acetone  by  a  special 
destructive  distillation  process.  Many  organic  compounds,  such  as 
ethyl  butyrate,  etc.,  were  recovered  from  the  fermented  kelp  liquor 
as  valuable  by-products.  The  Hercules  plant  at  one  tune  represented 
a  $5,000,000  investment  and  produced  about  24  tons  of  acetic  acid  or 
its  equivalent  in  acetone  per  day  from  1500  tons  of  kelp. 

*  See  H.  Hibbert,  Chemical  and  Metallurgical  Engineering,  1919,  pp.  397-400. 
f  Boll.  Assoc.  Ind.  Zucch.  e  Alcool,  Bologna,  1917.      142-145. 


SPECIAL   INDUSTRIAL   FERMENTED    PRODUCTS  651 

The  plant  has  since  been  closed,  as  have  others  of  a  similar  nature, 
which  operated  during  the  war  in  acetone  manufacture. 

LACTIC  ACID* 

Lactic  acid  is  used  in  large  quantities  in  the  tanning  industry  for 
deliming  and  plumping  the  heavier  hides.  It  also  finds  some  appli- 
cation in  other  industries  as  well.  There  is,  therefore,  a  moderate 
demand  for  the  acid  in  commerce. 

Several  raw  materials  have  been  used  in  its  preparation.  The 
waste  liquor  from  concrete  beet  silos  carries  about  2  per  cent,  of  this 
acid  and  has  been  used  direct  in  tanning  or  the  liquid  treated  to  recover 
the  lactic  acid  itself.  Amylaceous  substances  such  as  corn  starch, 
barley  malt,  etc.,  are  the  usual  raw  materials,  although  waste  milk 
from  creameries  or  the  whey  from  cheese  factories  may  also  be  employed 
to  advantage. 

If  starchy  materials  hydrolyzed  with  either  acid  or  malt  are  used,  it. 
is  possible  to  carry  out  lactic  fermentation  at  50°  or  higher  and  thus 
effectively  eliminate  the  butyric  organisms,  which  constitute  the 
principal  source  of  trouble.  If  milk  or  whey  is  the  culture  medium, 
B.  bidgaricus  or  other  vigorous  lactic  organism  adapted  to  milk  may  be 
used. 

The  fermentation  is  carried  out  in  the  presence  of  chalk  (CaCO3) 
or  zinc  carbonate.  Either  calcium  or  zinc  lactate  is  formed.  The 
zinc  lactate  crystallizes  satisfactorily  on  concentration;  calcium  lactate 
is  more  soluble.  The  salt  is  next  decomposed  with  sulphuric  acid  and 
distilled,  preferably  in  vacuo,  in  apparatus  not  corroded  by  lactic  acid 
vapors. 

Among  the  various  processes  described  in  the  literature,  the  Fried- 
bergerf  method  appears  to  possess  merit  although  it  is  difficult  to  see 
in  what  way  his  process  is  novel  to  the  extent  of  being  patentable. 
He  uses  a  pure  culture  of  B.  delbruckii  from  a  maltose  nutrient  medium 
which  is  transferred  to  sterile  dextrose  nutrient  medium  containing 
asparagin  and  peptone.  When  it  has  become  accustomed  to  the 
dextrose,  the  culture  is  grown  in  a  sterilized  medium  and  the  starter  so 
produced  used  to  ferment  a  sterilized  liquid  made  by  hydrolysis  of  any 
starchy  material  with  hydrochloric  acid.  The  fermentation  takes 

*  Prepared  by  W.  V.  Cruess. 

t  English  Patent  2507,  January  31,  1917. 


652  MICROBIOLOGY   OF   SPECIAL   INDUSTRIES 

place  in  the  presence  of  sterile  chalk.  Near  the  end  of  fermentation  the 
temperature  may  be  lowered  and  cultures  of  B.  bulgaricus  may  be 
added.  It  is  claimed  that  liquids  fermented  in  this  way  are  of  sweet 
odor  and  free  from  butyric  acid.  The  liquid  is  treated  with  0.3  to  0.5 
per  cent,  tannin,  calculated  on  the  weight  of  carbohydrates  originally 
present.  The  tannin  unites  with  the  albumins  of  the  liquid  resulting 
in  clarification.  The  clarified  liquid  is  filtered  and  decomposed  with 
sulphuric  acid  in  the  usual  way. 

Wehmer*  reports  that  15  grams  of  glucose  will  yield  under  average 
commercial  conditions  about  10.5  grams  of  lactic  acid;  whereas  the 
theoretical  yield  is  15  grams  according  to  the  following  equation: 

C6H1206  =  2C2H4OHCO2H. 
The  reactions  involved  in  the  purification  of  the  acid  are: 

2C2H4OHC02H  +  CaCO3  =  Ca(C2H4OHCO2)2  +  CO2  +  H2O. 

Ca(C2H,OHCO2)2  +  H2SO4  =  CaSO4  +  2C2H4OHCO2H. 
The  CaSO4  separates  as  a  precipitate. 

Wehmer  has  held  lactic  bacteria  for  six  years  in  calcium  lactate 
formed  during  fermentation  and  suggests  this  method  as  a  commercial 
means  of  storing  starters  of  desirable  strains  of  the  organism. 

Hexamethylene  tetraminf  has  been  used  instead  of  calcium  carbon- 
ate to  neutralize  the  lactic  acid  slowly  during  fermentation  and  the 
sugar  remaining  is  fermented  with  yeast  for  alcohol  production. 

Milk  and  whey  should  be  sterilized  before  use  and  fermented  with 
pure  cultures  of  lactic  organisms  to  minimize  the  constant  danger  from 
butyric  acid  fermentation  which  is  favored  by  neutralization  of  the 
liquid  with  calcium  carbonate. 

CITRIC  ACIDJ: 

Most  of  the  citric  acid  of  the  world's  commerce  is  obtained  from 
lemons,  although  it  is  possible  to  convert  various  sugars  into  citric  acid 
by  fermentation  processes. 

In  California  citric  acid  is  made  from  cull  lemons  from  the  packing 
houses.  Lemons  are  graded  very  closely  during  packing  in  order  that 
the  quality  of  the  packed  fruit  shall  be  uniform  and  the  fruit  attractive 
in  appearance.  This  results  in  a  large  percentage  of  culls  which  until 

*  Jour.  Soc.  Chem.  Ind.,  1906,  page  112. 

t  A.  Pollak,  U.  S.  Patent  1123920,   January,  1915. 

t  Prepared  by  W.  V,  Cruess. 


SPECIAL   INDUSTRIAL   FERMENTED    PRODUCTS  653 

recently  were  largely  an  economic  loss.  Through  the  investigations 
of  Dr.  E.  M.  Chace  and  C.  P.  Wilson  of  the  United  States  Bureau  of 
Chemistry,  a  commercially  stable  citric  acid  industry  has  been  founded 
in  California.  The  plant  of  the  Exchange  By-Products  Company 
at  Corona  is  capable  of  caring  for  over  100  tons  of  fruit  per  day. 
The  lemons  are  first  peeled  in  special  rotary  grating  machines.  The 
peels  are  distilled  to  recover  the  essential  oil.  The  peeled  fruit  is 
crushed  and  pressed  in  powerful  continuous  presses.  The  juice  is 
pumped  into  vats  of  about  30,000  gallons  each  where  it  is  permitted 
to  undergo  spontaneous  alcoholic  fermentation.  The  purpose  of  the 
fermentation  is  to  destroy  the  slimy  nature  of  the  juice,  making  it 
possible  to  filter  the  fermented  juice  easily.  The  fermented  juice  is 
filter  pressed;  the  clear  liquid  is  neutralized  with  calcium  carbonate 
at  the  boiling  point  because  calcium  citrate  is  more  insoluble  in  hot 
than  in  cold  liquids;  the  calcium  citrate  is  removed  by  filtration,  treated 
with  sulphuric  acid  in  slight  excess,  the  calcium  sulphate  is  removed 
by  filtration,  the  citric  acid  filtrate  is  partially  concentrated  in  open 
vats  by  a  stream  of  air,  concentrated  to  the  crystallizing  point  in 
glass-lined  vacuum  pans,  is  allowed  to  crystallize  in  shallow  vats, 
the  crude  crystals  are  redissolved  in  water,  and  recrystallized  to  give 
citric  acid  of  commerce.  The  manufacture  of  this  product  has  done 
much  to  stabilize  the  lemon  growing  industry  in  California. 

It  is  stated  that  it  is  possible  by  means  of  pure  cultures  of  certain 
varieties  of  citromyces  (a  penicillium-like  mold)  if  a  5  per  cent,  sugar 
solution  is  used,  to  convert  50  per  cent,  of  maltose,  30  per  cent,  of 
sucrose,  5  per  cent,  of  arabinose,  and  24  to  29  per  cent,  of  glycerol  into 
citric  acid.  Wehmer  states  that  citromyces  cultures  may  be  easily  ob- 
tained by  exposing  a  2  to  5  per  cent,  solution  of  citric  acid  and  cane 
sugar  to  the  air  for  several  weeks.  These  cultures  may  be  used  to 
convert  dilute  cane  sugar  solutions  containing  a  small  amount  of 
ammonium  nitrate,  dipotassium  hydrogen  phosphate  and  magnesium 
sulphate  into  dilute  solutions  of  citric  acid  which  may  be  safely  used  as 
lemonade  or  a  source  of  citric  acid.  As  high  as  a  4  per  cent,  citric 
acid  solution  may  be  readily  attained  at  18°  to  25°  in  8  to  14  days. 
If  left  too  long  the  acid  is  oxidized  and  the  liquid  in  time  becomes 
neutral  or  alkaline.  If  calcium  carbonate  is  added  before  inoculation, 
higher  yields  of  acid  are  obtained.  Above  25°  growth  is  inhibited  with 
many  strains  of  citromyces. 


654  MICROBIOLOGY   OF   SPECIAL  INDUSTRIES 

The  reaction  is  of  an  oxidizing  type  but  the  exact  set  of  chemical 
reactions  that  take  place  is  not  well  understood.  It  is  not  a  simple 
oxidation  process  because  citric  acid  possesses  a  branched  side  chain 
not  present  in  the  sugar  molecule.  For  each  50  grams  of  dextrose 
converted  into  13.3  grams  of  citric  acid,  10  liters  of  oxygen  is  required 
according  to  Wehmer. 

C.  citricus  has  given  very  high  yields  of  acid,  although  C.  Pfejferianus 
and  C.  Glaber  have  also  given  good  results.  Sterigmatocystis  nigra 
also  forms  citric  acid  in  dilute  sugar  solutions.  Many  forms  have 
been  studied  and  described  but  in  practically  all  cases  other  acids  and 
compounds  than  citric  acid  are  formed,  making  the  recovery  and 
purification  of  the  citric  acid  very  difficult  or  impossible. 

Most  of  the  citromyces  that  have  come  to  the  writer's  attention 
form  during  the  first  stages  of  growth  a  cottony  white  mycelium. 
This  usually  later  turns  to  pale  green  and  in  time  olive  brown;  some 
cultures  remain  permanently  white.  Citromyces  may  readily  be 
mistaken  for  Penicillium  expansum,  an  organism  which  tends  to  con- 
taminate cultures  of  citromyces  if  precautions  are  not  taken. 

It  is  possible  that  the  citromyces  fermentation  may  in  time  be  used 
for  commercial  purposes,  but  it  is  at  the  present  time  in  the  experimental 
stage. 

WHITE  LEAD* 

Basic  acetate  of  lead  used  so  extensively  in  paint  is  of  finer  grain 
and  better  covering  quality  if  made  by  the  Dutch  or  fermentation  proc- 
ess than  if  made  by  purely  chemical  processes.  Grids  of  pure  lead 
are  stacked  between  tiers  of  spent  tan  bark;  several  layers  of  lead 
and  tan  bark  being  built  up  in  well-insulated  rooms.  Acetic  acid 
and  CO2  are  formed.  The  acid  fumes  rise  from  the  heat  of  fermenta- 
tion and  combine  with  the  lead  plates,  forming  a  crust  of  white  lead. 
Most  of  the  lead  is  converted  to  the  basic  acetate. 

The  exact  nature  of  the  fermentation  is  not  well  understood.  It 
is  probably  not  of  the  usual  "alcohol-acetic"  type  carried  out  by  S. 
ellipsoideus  or  S.  cerevisia  and  Bact.  aceti,  but  is  possibly  a  mixed  fer- 
mentation of  several  types  of  bacteria.  Various  substitutes  for  tan 
bark  have  been  tried  but  Calif ornian  manufacturers  at  least  have  found 
the  spent  bark  by  far  the  most  suitable  material, 

*  Prepared  by  W.  V,  Cruess. 


SPECIAL   INDUSTRIAL   FERMENTED   PRODUCTS  655 

LEATHER* 

In  the  manufacture  of  leather,  bacteria  play  a  very  important  and 
extremely  interesting  role.  Success  depends  to  a  very  great  degree 
upon  the  proper  control  of  microorganisms  during  the  various  steps 
of  the  manufacturing  process. 

Most  hides  and  skins  are  received  at  the  tannery  fresh  with  more  or 
less  adhering  blood  and  flesh.  Hides  from  outlying  districts  or  foreign 
countries  are  received  in  the  salted  or  dried  states. 

The  hides  are  first  placed  in  water,  in  the  so-called  "soaking  pits," 
to  remove  the  blood  from  fresh  hides,  the  salt  from  salted  hides  and  to 
plump  and  soften  the  dried  hides.  Formerly  "putrid  soaks"  were 
used.  The  liquid  in  these  soaks  had  stood  for  a  long  enough  period  to 
become  swarming  with  putrefactive  organisms.  Much  of  the  gelatin 
of  the  hide  was  dissolved  and  often  the  grain  of  the  hide  was  injured 
by  bacterial  action.  B.  fluorescens  liquefaciens,  B.  megatherium, 
B.  subtilis,  B.  proteus  vulgaris,  B.  proteus  mirabilis,  -were  commonly 
found  in  this  liquid  according  to  J.  T.  Wood.f  None  are  beneficial  to 
the  hide  and  most  of  them  are  harmful.  If  the  water  is  changed  fre- 
quently there  is  less  danger  of  putrefaction;  Proctor  recommends  the 
use  of  an  antiseptic  solution  of  1:1000  sodium  hydroxide  or  from  i  to 
3  per  1000  of  sodium  sulphide  to  prevent  bacterial  growth  during 
soaking. 

The  wool  is  often  removed  from  sheep  skins  by  bacterial  action. 
The  hides  are  hung  on  racks  in  an  air-tight  room  at  15°  to  20°.  Wood 
reports  the  following  organisms  to  be  active  during  this  "sweating" 
process:  B.  fluorescens  liquefaciens,  Bad.  pilline,  and  a  streptococcus. 
These  organisms  secrete  enzymes  which  dissolve  the  cementing  sub- 
stance at  the  roots  of  the  wool  permitting  the  wool  to  be  easily  removed. 
It  is  difficult  to  check  or  control  this  process;  usually  some  hides  from 
the  "sweating  stove"  will  be  spoiled  by  too  prolonged  putrefaction. 
Caustic  sodium  sulphide  is  used  to  a  considerable  extent  to  replace  the 
sweating  process. 

The  hair  is  removed  from  hides  and  calf  skins  by  liming.  The 
hides  are  first  placed  in  a  lime  pit  consisting  of  water  and  slaked  lime 
that  has  been  in  use  for  some  time.  This  old  lime  pit  contains  proteo- 

*  Prepared  by  W.  V.  Cruess. 

t  Wood,  J.  T:  Bacteriology  of  the  Tanning  Industry,  Journal  of  Soc.  Chem.  Ind.,  1910,  666. 


656  MICROBIOLOGY   OF    SPECIAL   INDUSTRIES 

lytic  bacteria  which  develop  upon  the  gelatin  and  other  nutrient  mate- 
rial dissolved  from  the  hides  previously  treated.  These  organisms  exert 
an  appreciable  depilatory  action  and  considerably  increase  the  activity 
of  the  lime.  Some  of  this  effect  is  due  to  ammonium  salts  and  amines, 
formed  by  the  bacteria.  Much  of  the  bacterial  effect  can  be  duplicated 
by  adding  ammonium  sulphate.  From  this  lime  pit  hides  in  time  go  to 
pits  containing  new  lime  and  thence  to  the  beam  where  the  hair  is  re- 
moved by  scraping. 

If  the  hide  is  for  sole  leather  or  other  heavy  leather  it  goes  direct 
to  a  dilute  lactic  acid  or  dilute  mineral  acid  bath  where  the  excess  lime 
is  dissolved  without  removal  of  any  appreciable  amount  of  the  hide 
substance  needed  to  give  rigidity  to  the  leather. 

Soft  leathers  require  a  different  treatment.  Fine  leathers  such  as 
glove  leather  are  usually  given  a  puering  treatment  in  which  the  skins 
are  placed  in  a  dilute  infusion  of  dog  dung  which  has  previously  been 
permitted  to  stand  several  days  to  develop  the  proper  types  of  bac- 
teria. Other  skins  are  given  a  bating  process  in  pigeon  or  hen  dung 
infusion  or  in  a  proprietary  bating  solution.  The  hen  or  pigeon 
dung  is  prepared  for  use  by  soaking  in  warm  water  several  days.  Fer- 
mentation and  vigorous  development  of  bacteria  ensue.  The  ferment- 
ing infusion  is  diluted  with  water  and  mixed  with  the  skins  in  the 
"bating  wheel"  which  consists  of  a  large  wooden  paddle  wheel  revolv- 
ing in  a  tank  of  the  bating  liquor.  In  the  puer  and  bate  liquors  the 
lime  is  dissolved  from  the  hides  to  some  extent  by  lactic  acid  and  also 
through  the  action  of  amines  and  ammonium  salts  formed  by  the  bac- 
teria from  proteins  of  the  skins.  Some  of  the  intra-cellular  substance 
of  the  skin  is  dissolved.  The  skins  become  soft  and  pliable,  i.e.,  "fall. " 
The  surface  becomes  slippery  and  the  skin  retains  the  imprint  of  the 
finger  if  pressed  between  the  thumb  and  ringer.  Too  prolonged  bating 
or  puering  results  in  pitting  of  the  hide;  in  fact,  it  is  possible  to  cause 
the  hide  to  go  completely  into  solution  by  several  days'  bating  at 
37°,  the  temperature  ordinarily  used  in  practice. 

Many  attempts  have  been  made  to  replace  the  dung  infusions  with 
pure  cultures.  In  cooperation  with  F.  H.  Wilson  the  writer  isolated  a 
number  of  organisms  from  bate  liquors.  Most  of  these  were  of  the 
colon  group;  some  of  the  proteus  group.  The  colon  group  of  organisms 
gave  good  results  in  pure  culture  when  grown  in  dilute  milk  (diluted 
i :  10)  with  water  or  in  dilute  sugar  solutions.  The  milk  or  other  sugars 


SPECIAL  INDUSTRIAL   FERMENTED   PRODUCTS  657 

used  protected  the  skins  against  overbating  and  injury  but  at  the 
same  time  permitted  effective  removal  of  the  lime  and  very  satisfactory 
bating  or  softening  of  the  texture.  Similarly,  good  results  were  ob- 
tained by  Noble  at  the  University  of  California  by  using  pure  cultures 
of  B.  subtilis  in  hay  infusions  or  other  cheap  and  suitable  liquids. 
The  practice  which  he  preferred  was  to  heat  an  infusion  of  hay  to  boiling 
and  to  allow  this  to  stand  until  a  good  growth  of  B.  subtilis  had 
developed. 

Patented  mixtures  of  a  pure  culture  of  Bacillus  erodiens  or  other 
suitable  organisms  and  a  suitable  nutrient  medium  in  dry  and  soluble 
form  have  been  successfully  employed  to  replace  dung  bates.  A  mix- 
ture of  pancreas  extract  and  ammonium  sulphate  has  been  sold  under 
a  trade  name  and  used  with  fair  success.  It  therefore  appears  that 
the  use  of  the  dung  infusions  is  not  essential. 

At  one  time  the  skins  from  the  bating  liquor  were  transferred  to 
a  fermenting  infusion  of  bran  known  as  the  " drench"  for  18  to  24 
hours.  Acid  formed  by  B.  jur juris  and  similar  organisms  removed  the 
last  traces  of  lime.  At  the  present  time  a  " pickle"  consisting  of  dilute 
sulphuric  acid  and  sodium  chloride  is  commonly  used  to  remove  the 
lime  left  in  the  skins  from  the  bate  liquor.  The  calcium  sulphate  so 
formed  crystallizes  in  the  pickle  vats  as  a  hard  incrustation. 

The  skins  are  now  ready  for  the  tan  pits.  They  enter  the  old  and 
more  dilute  tan  liqu'ors  first.  In  these  the  bacteria  carried  over  on  the 
hides  from  the  bate,  etc.,  adapt  themselves.  Some  lactic  acid  is  formed 
and  tends  to  plump  the  skins  and  facilitate  the  penetration  of  the  tannin. 
Dilute  tannic  acid  solutions  must  be  used  at  first  to  permit  deep  pene- 
tration of  the  tannin.  The  concentration  of  tannin  is  increased  in 
the  succeeding  pits  until  a  saturated  solution  is  reached.  The  extrac- 
tive matter  from  the  tan  bark  in  the  dilute  liquors  supports  a  varied 
growth  of  lactic  bacteria,  yeasts  and  molds,  while  sufficient  tannin 
is  present  to  check  effectively  the  growth  of  putrefactive  organisms. 
Tan  bark  is  used  for  ordinary  leather  while  sumach  and  other  light 
colored  tannin  extracts  are  used  for  lighter  colored  leather. 

Tannin  may  be  replaced  by  chrome  alum  or  potash  alum,  or  other 
mineral  tanning  materials.  Some  of  the  toughest  and  most  resistant 
leathers  are  of  this  type. 


42 


658  MICROBIOLOGY   OF    SPECIAL   INDUSTRIES 

INDIGO  * 

Indigo  is  now  for  the  most  part  made  synthetically.  This  dye  was 
formerly  made  from  certain  species  of  Indigo/era,  principally  /.  tinctoria. 
This  plant  contains  a  glucoside,  indican,  which  by  fermentation  and 
oxidation  yields  indigo. 

The  plants  are  placed  in  water  at  a  temperature  of  25°  to  35°  and 
undergo  a  spontaneous  alkaline  fermentation  which  splits  up  the  indican 
into  a  sugar  (indigluciri)  and  indigo  white  which  remain  in  the  solution. 
This  solution  is  then  thoroughly  aerated  and  the  indigo  white  oxidized 
into  indigo  blue  which  is  insoluble  and  forms  a  sediment.  This  sedi- 
ment is  dried  and  constitutes  the  old  indigo  of  commerce. 

Many  bacteria  are  found  in  the  fermenting  liquid,  but  the  cause 
of  transformation  has  been  shown  to  be  a  specific  form,  Bacillus  indigo- 
genus,  closely  related  to  Friedlander's  pneumonia  bacillus.  It  is 
strongly  aerobic  and  surrounded  by  a  gelatinous  envelope. 

RETTING* 

The  separation  of  the  fibers  of  flax,  hemp,  ramie  and  similar  plants 
is  brought  about  by  a  complex  spontaneous  fermentation.  The  plants 
are  either  left  on  the  surface  of  grassy  meadows  exposed  to  alternate 
wetting  and  drying  or  immersed  in  water.  In  either  case,  the  tissues 
are  gradually  disintegrated  by  microbial  action,  more  rapidly  in  the 
wet  process. 

The  fermentation,  principally  bacterial,  is  due  to  many  species. 
Several  have  been  described  as  being  the  principal  agent  in  the  process 
but  it  is  probable  that  the  effects  are  due  to  the  united  action  of  several, 
both  aerobic  and  anaerobic. 

Among  the  forms  to  which  the  retting  has  been  attributed  are  B. 
amylobacter  of  van  Tieghem,  an  anaerobic  form  which  attacks  the  pec- 
tic  matters  and  to  some  extent  the  cellulose.  Granulobacter  pectino- 
vorum  of  Beyerinck  and  van  Delden,  also  anaerobic,  transforms  the 
pectic  matters  into  sugars  which  it  decomposes,  producing  butyric 
acid.  Many  other  forms  have  been  described  and  part  of  the  work  has 
been  ascribed  to  Mucor,  Penicillium,  and  various  molds. 

Cultures  of  Granulobacter  pectinowrum  and  other  forms  have  been 
successfully  used  to  hasten  the  process. 

*  Prepared  by  F.  T.  Bioletti. 


DIVISION  VIII 

MICROBIOLOGY  OF  DISEASES  OF  MAN  AND  DOMESTIC  ANIMALS 

CHAPTER  I* 

METHODS  AND  CHANNELS  OF  INFECTION 
INFECTION  DEFINED 

The  term  infection  implies  the  entrance  of  animal  or  vegetable 
organisms  into  the  body  of  another  animal  or  plant,  their  multiplica- 
tion and  their  injury  to  that  body.  In  most  instances  the  organisms 
enter  the  tissues  of  the  animal  or  plant  body,  although  this  is  not  true 
in  every  case  of  infection.  It  is  possible  in  certain  instances  to  produce 
the  symptoms  of  an  infection  by  introducing  into  the  body  the 
chemical  products  elaborated  by  some  pathogenic  organisms.  For 
example,  the  injection  of  tetanus  toxin  into  the  body  causes  the  typical 
symptoms  of  tetanus  to  result.  Tetanus  toxin  is  made  by  growing 
B.  tetani  in  beef  broth  under  anaerobic  conditions  and  filtering  out  the 
bacteria  by  passing  through  porcelain  filters.  These  chemical  prod- 
ucts do  not  occur  naturally  unassociated  with  the  pathogenic 
organisms  and  therefore  they  do  not  produce  infections  when  artifi- 
cially injected  in  the  usual  sense. 

The  disease-producing  organisms  with  which  we  will  especially 
concern  ourselves  in  the  subsequent  discussion  are  those  which  are  very 
minute  in  size  and  are  of  three  kinds:  first,  bacteria;  second,  protozoa; 
and  third,  ultramicroscopic  microorganisms  or  viruses. 

It  is  essential  to  have  clearly  in  mind  what  is  meant  by  an  infectious 
disease  and  a  contagious  disease  before  entering  into  any  detailed  dis- 
cussion, although  some  authorities  attempt  to  make  no  distinction. 
An  infectious  disease  is  any  disease  produced  in  the  plant  or 
animal  body  which  is  due  to  a  foreign  animal  or  plant  organism.  The 
name  is  applied  to  the  nature  of  the  cause  of  the  disease.  A  contagious 

*  Prepared  by  E.  F.  McCampbell. 

659 


66o   MICROBIOLOGY   OF  DISEASES   OF. MAN  AND  DOMESTIC  ANIMALS 

disease  is  an  infectious  disease  which  is  transmitted  from  one  individual 
to  another  by  contact.  The  term  refers  to  the  method  of  transmission 
rather  than  to  the  cause  of  the  disease.  It  is  possible  that  certain  conta- 
gious diseases  may  be  transmitted  by  indirect  contact  or  by  the  agency 
of  fomites  but  many  authorities  now  hold  the  view  that  these  factors 
are  non-essential  and  that  most  contagious  diseases  are  transmitted  by 
direct  contact. 

MICROORGANISMS  OF  DISEASE  CONSIDERED  AND  CLASSIFIED 

PATHOGENIC  BACTERIA. — Bacteria  which  produce  disease  are  known 
as  pathogenic  bacteria.  Of  the  many  thousand  species  of  bacteria  only 
a  comparatively  few  species  have  anything  to  do  with  the  diseased 
processes  in  the  plant  or  animal  body.  Those  bacteria  which  are 
capable  of  growing  in  the  body  of  animal  or  plant  may  be  designated  as 
parasitic  bacteria.  Some  bacteria  can  grow  only  in  the  animal  or  plant 
body  and  do  not  exist  for  any  period  of  time  outside  of  it.  They  are 
known  as  obligate  parasites.  There  are  others  which  may  produce 
disease  in  the  animal  or  plant  body  which  can  grow  and  reproduce 
outside  the  body.  They  are  known  as  facultative  saprophytes.  There 
are  still  other  bacteria  which  ordinarily  live  outside  the  animal  and 
plant  body  and  which  exist  largely  upon  dead  organic  material,  which 
when  taken  into  the  body  occasionally  produces  disease  processes. 
They  are  called  facultative  parasites.  As  an  example  of  an  obligate 
parasite  the  Bact.  lepra  of  leprosy  may  be  cited,  although  in  this 
instance  certain  observers  have  claimed  to  have  cultivated  the  bacillus 
in  pure  culture.  However,  the  results  are  not  in  any  sense  uniform. 
Improved  bacteriological  technic  has  made  possible  the  cultivation  of  a 
large  number  of  bacteria  which  heretofore  were  regarded  as  obligate 
parasites.  As  examples  of  facultative  saprophytes  the  B.  typhosus  of 
typhoid  fever  and  the  Msp.  comma  of  cholera  may  be  mentioned. 
As  examples  of  facultative  parasites  B.  tetani  of  tetanus  and  Bact. 
wclchii  of  gaseous  gangrene  may  be  mentioned. 

PATHOGENIC  PROTOZOA. — There  are  several  infectious  diseases  in 
man  and  animals  which  are  caused  by  pathogenic  protozoa.  Among 
the  common  diseases  due  to  protozoa  there  may  be  mentioned  malaria, 
syphilis,  rabies  (the  nature  of  the  organisms  involved  in  syphilis  and 
rabies  is  not  well  understood  however),  amoebic  dysentery,  Texas  fever, 
infectious  jaundice  of  dogs,  and  the  various  trypanosome  infections 


METHODS    AND    CHANNELS    OF   INFECTION  66 1 

such  as  sleeping  sickness,  nagana,  dourine,  and  mal  de  caderas.  It  is 
difficult  to  cultivate  artificially  the  pathogenic  protozoa  outside  the 
animal  body  in  pure  culture.  The  Trypanosoma  brucei  of  nagana  and 
the  Trypanosoma  lewisi  of  the  rat  have  been  cultivated.  The 
Entamceba  coli  and  the  Entamozba  tetragena  of  dysentery,  the  various 
types  of  the  Plasmodittm  malaria,  and  the  Treponema  pallidum  of 
syphilis  have  also  been  cultivated,  and  it  is  stated  that  under  certain 
conditions  the  Piroplasma  bigeminum  of  Texas  fever  may  be  artificially 
grown. 

ULTRAMICROSCOPIC  MICROORGANISMS  OR  VIRUSES. — There  are  some 
infectious  diseases  the  causes  of  which  have  never  been  discovered. 
The  infectious  agents  in  most  instances  cannot  be  cultivated  and 
cannot  be  stained  by  the  ordinary  bacteriological  methods.  The 
presence  of  ultramicroscopic  organisms  has  been  demonstrated  in 
several  ways.  For  example,  when  the  ordinary  bacterial  culture  is 
run  through  a  fine  porcelain  filter,  the  filtrate  contains  no  microorgan- 
isms and  consequently  when  inoculated  into  animals  is  non-infectious, 
although  if  soluble  toxins  be  present  there  may  be  evidences  of  an 
intoxication.  When  the  viruses  or  the  infected  body  fluids  of  men  or 
animals  suffering  from  the  diseases  mentioned  below  are  passed  through 
a  fine  porcelain  filter  the  filtrate  remains  infectious,  therefore  demon- 
strating that  the  viruses  or  microorganisms  are  filtrable  and  are  prob- 
ably so  small  that  they  cannot  be  seen.  Examples  of  diseases  due  to 
agents  belonging  to  this  class  are  as  follows:  hog  cholera,  yellow  fever, 
foot-and-mouth  disease,  rinderpest,  epithelioma  contagiosum  of  fowls, 
chicken  typhus,  horse  sickness,  acute  poliomyelitis,  etc.  There  are 
several  infectious  diseases  of  unknown  cause,  the  viruses  of  which  are 
not  filtrable;  for  example,  smallpox,  cowpox  and  vaccinia,  typhus  fever 
and  Rocky  Mountain  spotted  fever.  There  are  still  other  diseases  of 
unknown  cause  about  which  nothing  is  known  regarding  the  filterability 
of  the  etiological  agents  of  the  disease.  Scarlet  fever,  chickenpox  and 
measles  belong  to  this  class.  These  diseases  .can  be  inoculated  into 
animals  only  with  great  difficulty  and  the  virus  cannot  be  cultivated 
or  secured  in  sufficient  quantities  from  the  experimental  animals  for 
study.  A  possible  explanation  of  some  of  these  diseases  of  unknown 
cause  may  be  found  in  the  proposition  that  two  microorganisms  may 
each  produce  non-toxic  substances,  and  that  when  these  non-toxic 
substances  come  together,  a  toxic  substance  may  be  produced.  This 


662    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

condition  of  affairs  might  explain  certain  infectious  diseases  in  which 
microorganisms  are  known  to  occur,  and  in  which  they  cannot  be 
directly  connected  with  the  disease  as  causative  factors.  For  example, 
the  Strept.  pyogenes  very  frequently  occurs  in  both  scarlet  fever  and 
smallpox.  It  has  been  shown  absolutely  that  this  organism  is  not  the 
cause  of  these  diseases,  but  there  is  a  remote  possibility  that  it  may 
act  in  the  so-called  associative  relation  with  some  other  microorganism 
or  virus,  as  mentioned  above,  and  produce  the  typical  symptoms  of 
these  diseases.  It  has  been  recently  stated  that  scarlet  fever  is 
due  to  a  filtrable  virus  but  there  is  every  reason  to  believe  that  the 
occurrence  of  the  Strept.  pyogenes  materially  changes  the  character  of 
the  infection  and  makes  it  more  severe.  The  associative  relationship 
of  infectious  organisms  is  probably  not  the  logical  explanation  for  all 
infections  of  this  character.  It  might  be  mentioned  in  this  connection 
that  the  view  is  held  by  some  investigators  that  some  of  the  infectious 
diseases  of  unknown  etiology  are  due  to  enzymes  and  that  a  so-called 
autocatalysis  explains  the  seeming  reproduction  in  the  body  of  the 
viruses.  This  theory  is,  however,  without  substantial"  proof. 

THE  DISTRIBUTION  OF  PATHOGENIC  MICROBIC  AGENTS  IN  NATURE 

The  causal  microorganisms  of  most  of  our  infectious  diseases  are 
found  principally  in  the  bodies  of  diseased  man  and  animals.  There  are 
some  exceptions  to  their  being  found  only  in  the  bodies  of  the  diseased. 
Notable  examples  are  found  among  certain  of  the  wild  animals  such  as 
the  brush-buck,  wildebeast  and  others  which  serve  as  reservoirs  for 
the  microorganisms  of  some  of  the  most  fatal  of  protozoal  diseases. 
These  animals  seem  to  be  naturally  immune.  Various  insects  which 
are  factors  in  the  transmission  of  certain  infectious  diseases  do  not  suffer 
from  these  diseases  in  any  form  and  are  naturally  immune.  The  most 
common  source,  however,  is  the  diseased  animal  or  human  body. 
There  is  no  doubt,  for  example,  that  the  natural  habitat  of  the  Bad. 
diphtheria  is  in  the  throat  and  nasal  passages  of  persons  suffering  from 
or  convalescing  from  diphtheria.  Occasionally  these  bacteria  are  also 
found  in  the  nasal  passages  and  throats  of  persons  who  have  never  had 
diphtheria.  The  same  is  true  of  the  M .  intracellularis  var.  meningitidis, 
of  cerebro-spinal  fever.  The  B.  typhosus  of  typhoid  fever  also  has  its 
natural  abode  in  the  intestinal  tract  of  persons  suffering  from  or  con- 
valescing from  the  fever.  The  same  is  true  with  the  majority  of  the 


METHODS   AND    CHANNELS    OF   INFECTION  663 

causal  microorganisms.  There  are  some  microbic  agents,  however, 
which  exist  in  the  soil  but  probably  do  not  undergo  multiplication  such 
as  the  B.  tetani  of  tetanus  or  lockjaw,  Bact.  Welchii  of  emphysematous 
or  gaseous  gangrene,  and  the  B.  botulinus  of  meat  poisoning.  These 
bacteria  sometimes  exist  in  the  intestinal  tracts  of  animals  such  as  the 
horse  and  in  all  probability  their  occurrence  in  the  soil  is  due  to  their 
deposition  in  manure. 

THE  OCCURRENCE  OF  PATHOGENIC  MICROBIC  AGENTS  UPON  AND  IN 
THE  BODIES  OF  HEALTHY  ANIMALS  AND  MAN 

The  exposure  to  the  air  of  the  external  surfaces  of  the  body,  of 
course,  makes  it  especially  easy  for  microorganisms  to  collect  upon 
them.  The  large  percentage  of  the  microorganisms  which  collect  on 
the  external  surfaces  are  non-pathogenic  but  there  are  frequently  dis- 
ease-producing ones  among  them.  The  various  varieties  of  the  M. 
pyogenes  are  almost  universally  present  on  the  skin  and  also  on  the 
exposed  mucous  membranes.  Strept.  pyogenes,  Bact.  influenzae,  Bact. 
tuberculosis,  M.  intracellularis  var.  meningitidis,  Strept.  pneumonia, 
Bact.  diphtheria  and  many  other  species  may  be  present.  The  mouth 
and  nose  are  excellent  places  for  microorganisms  to  collect  and  excellent 
for  their  growth  as  the  requisite  conditions  such  as  food,  heat  and 
moisture  are  present.  It  has  been  stated  on  competent  authority  that 
all  the  species  of  bacteria  which  have  been  described  as  occurring  in 
various  parts  of  the  body  have  also  been  found  in  the  mouth.  These 
bacteria  do  not  necessarily  produce  disease  or  injure  the  body  unless 
the  vitality  is  lowered  and  they  enter  into  the  tissues.  They  feed  upon 
the  desquamating  cells  and  the  excretions.  It  is  exceedingly  interest- 
ing to  note  that  Bact.  tuberculosis  and  Bact.  diphtheria,  as  before  stated, 
have  been  found  in  the  nose  of  persons  who  have  never  had  these  dis- 
eases. These  bacteria  have  also  been  shown  to  be  virulent  and 
undoubtedly  such  persons  are  extremely  dangerous  to  other  more 
susceptible  persons.  It  is  also  frequently  noted  that .  pathogens  are 
found  in  the  bodies  of  persons  after  they  have  recovered  from  the  dis- 
ease and  that  these  individuals  disseminate  the  microorganisms  and 
infect  non-immune  individuals.  This  may  be  the  case  in  diphtheria, 
epidemic  meningitis,  typhoid,  Asiatic  cholera  and  dysentery  "bacillus 


664   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

In  regard  to  the  occurrence  of  microbic  agents  in  the  internal  organs 
of  the  body  the  following  may  be  said.  For  a  long  time  it  was  claimed 
that  the  internal  organs  of  man  and  animals  were  sterile.  Neisser  is 
one  authority  for  the  statement  that  the  internal  organs  of  healthy 
animals  are  sterile.  This  has  been  shown  not  to  be  the  case  universally. 
Experiments  have  shown  that  fifty  per  cent  of  the  internal  organs  of 
rabbits,  guinea-pigs,  cats,  dogs,  mice,  horses  and  cattle  are  not  sterile. 
Bact.  tuberculosis  has  been  found  in  absolutely  normal  human  and 
bovine  lymph  glands.  The  various  pus-producing  micrococci  have 
been  frequently  found  in  the  spleen,  kidney,  liver,  etc.  Perhaps  the 
commonest  group  of  bacteria  to  be  isolated  from  the  internal  organs  are 
the  intestinal  forms.  It  has  been  demonstrated  that  intestinal  micro- 
organisms invade  the  tissues  with  surprising  rapidity  when  for  any 
reason  the  resistance  of  the  body  is  lowered.  It  has  been  noted  also 
that  there  are  more  bacteria  in  the  internal  organs  of  animals  which 
have  been  fasted  than  in  those  which  have  been  fed.  Peristaltic  action 
and  the  diffusion  of  food  through  the  intestinal  wall  may  be  influencing 
factors.  The  fact  that  the  internal  organs  are  not  sterile  in  every  case 
is  important  as  it  may  account  for  the  so-called  autogenic  infections. 

THE  MANNER  IN  WHICH  INFECTIOUS  AGENTS  ENTER  THE  BODY  AND 

THEIR  SOURCES 

Air-borne  Infections. — The  causal  microorganisms  of  infectious 
diseases  are  frequently  excreted  from  the  body  of  the  diseased  individual 
and  are  deposited  on  the  clothing,  furnishings,  on  the  floors  and  walls, 
or  on  the  ground.  These  microorganisms  probably  do  not  proliferate 
except  in  rare  instances,  but  frequently  remain  virulent  for  a  short 
period  of  time  and  are  capable  of  being  carried  through  the  air  for  short 
distances,  producing  in  certain  instances  disease  in  other  individuals. 
There  is  no  doubt  that  in  diseases  such  as  smallpox,  measles,  scarlet 
fever  and  other  acute  exanthematous  diseases  together  with  such 
diseases  as  plague  and  diphtheria,  that  the  infectious  agents  may  be 
carried  through  the  air  after  having  been  deposited  on  clothing  and 
furnishings.  However,  recent  investigations  have  shown  that  this 
method  of  transferring  infection  is  comparatively  rare  and  that  most 
infections  are  transmitted  by  direct  contact. 

In  the  beginning  it  was  supposed  that  the  only  way  that  bacteria 
could  be  carried  in  the  air  was  after  having  been  dried  on  particles  of 


METHODS    AND    CHANNELS    OF    INFECTION  665 

dust  and  carried  by  currents  of  air.  This,  however,  has  been  shown 
not  always  to  be  the  case  and  we  now  know  that  infectious  micro- 
organisms may  be  carried  on  small  particles  or  droplets  of  sputum  or 
moisture.  These  two  types  of  aerial  infection  are  known,  respectively, 
as  dust  and  droplet  infection. 

Dust  Infection. — Infectious  microorganisms  to  remain  virulent  and 
be  able  to  produce  infection  must  be  able  to  successfully  resist  drying 
after  being  affixed  to  particles  of  dust.  After  being  dried  the  particles 
are  frequently  moved  and  whirled  about  by  air  currents.  The  larger 
particles  of  material  quickly  settle  down  but  the  small,  almost  invisible 
pieces  of  dried  material  may  remain  suspended  for  three  or  four  hours. 
Tt  is  these  small  particles  which  are  usually  inhaled  or  deposited  on  the 
skin  and  mucous  membranes  of  normal  individuals  that  produce  in- 
fections providing  the  microorganisms  have  not  been  killed  by  drying 
or  exposure  to  sunlight.  Bad.  tuberculosis  is  sometimes  carried  in  this 
way  as  well  as  certain  other  pathogens.  The  fact  that  small-pox  virus 
remains  active  after  drying  indicates,  at  least,  that  dust  containing 
it  may  be  infectious.  The  extent  of  such  dissemination  is  quite  limited. 

Droplet  Infections. — It  has  been  demonstrated  that  during  the  proc- 
esses of  talking,  coughing,  and  sneezing,  small  bubbles  or  droplets  of 
sputum  are  thrown  out  into  the  air.  These  particles  remain  suspended 
for  some  time  and  may  be  inhaled  or  deposited  elsewhere.  It  is  sur- 
prising the  distance  that  these  small  particles  may  be  carried.  It  is 
stated  that  they  are  frequently  thrown  out  thirty  feet  or  more.  It  has 
been  shown  that  Bact.  tuberculosis  is  rarely  thrown  out  over  four  or  five 
feet  by  the  cough  of  the  tuberculous  individual.  It  should  be  re- 
membered that  these  bacteria  will  remain  alive  two  to  three  weeks 
when  in  the  dark  but  that  they  live  only  a  few  hours  when  exposed 
to  the  sunlight.  The  pathogenic  microorganisms  or  viruses  which 
are  commonly  disseminated  by  droplets  of  moisture  are  those  of 
whooping  cough,  mumps,  measles,  influenza,  epidemic  meningitis, 
pneumonia,  and  pneumonic  plague. 

Air-borne  infections  rarely  occur,  as  previously  stated,  and  are  not  of 
great  importance  in  the  open  air  where  sunlight  has  free  access  to  the 
disease  germs  but  this  type  of  infection  sometimes  occurs  in  crowded 
quarters  such  as  dark  shops,  schools,  tenements  and  railway  trains. 
However,  the  factor  of  direct  contact  must  be  given  its  due  weight  in 
such  instances. 


666   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

Water-borne  Infections. — Pure  infections  of  this  type  occur  in  prac- 
tically only  five  diseases,  namely,  Asiatic  cholera,  typhoid  fever, 
paratyphoid  fever,  and  in  dysentery  of  the  amoebic  and  bacillary  forms. 
The  length  of  time  that  these  microorganisms  will  remain  alive  in  the 
water  depends  on  the  quantity  and  quality  of  organic  matter  present. 
Only  under  rare  circumstances  do  bacteria  proliferate  in  water  of 
very  high  organic  matter  content.  Ordinarily  microorganisms  will 
live  only  a  few  days  if  the  water  is  absolutely  pure.  They  have,  how- 
ever, been  known  to  live  for  several  weeks  in  ordinary  river  water.  The 
drinking  of  water  or  of  fluids  or  material  contaminated  by  water  is 
the  common  but  not  the  only  way  these  diseases  are  acquired. 

Infections  from  Soil. — The  soil  as  a  source  of  infectious  micro- 
organisms is  of  importance  in  only  a  few  diseases,  namely,  anthrax, 
tetanus,  symptomatic  anthrax,  malignant  edema,  emphysematous 
gangrene,  Asiatic  cholera,  and  typhoid  fever.  In  the  first  five  men- 
tioned infection  always  takes  place  through  some  wound  usually  in 
the  skin  and  in  the  last  two  diseases  mentioned  infection  is  usually 
through  the  intestinal  tract  but  may  also  occur  by  means  of  wounds. 
The  microorganisms  of  anthrax,  tetanus  and  emphysematous  gangrene, 
or  more  specifically  the  spores,  will  remain  in  soil  for  long  periods  of 
time.  They  are  sometimes  found  in  the  active  vegetative  stage  but 
it  is  probable  that  they  do  not  proliferate  to  any  extent  in  the  soil. 
They  exist  as  ordinary  saprophytes.  The  microorganisms  of  typhoid 
and  cholera  have  been  known  to  remain  alive  for  a  year  or  more  in 
soils  containing  large  quantities  of  organic  matter.  The  various 
pyogenic  micrococci  are  also  occasionally  found  in  the  soil  and  may 
enter  the  body  of  man  and  animals  through  wounds.  These  last- 
mentioned  organisms  may  live  for  indefinite  periods  of  time  on  the 
skin  and  enter  the  body  only  when  the  resistance  of  some  tissue  is 
lowered. 

Infection  from  Food. — Quite  a  large  variety  of  pathogenic  micro- 
organisms have  been  found  in  the  various  food  products.  Milk  is 
perhaps  the  most  common  food  product  to  be  infected.  The  causal 
agents  of  diphtheria,  scarlet  fever  and  some  other  diseases  have  been 
disseminated  by  means  of  milk.  Milk  contaminated  by  water  con- 
taining B.  typhosus  may  be  the  means  of  conveying  typhoid  fever,  and 
the  dissemination  of  Malta  fever  is  accomplished  by  the  drinking  of  the 
milk  of  infected  goats.  Typhoid  fever  has  also  been  known  to  have 


METHODS   AND    CHANNELS    OF   INFECTION  667 

been  acquired  from  the  eating  of  vegetables  which  have  been  washed  in 
water  containing  the  pathogens.  Oysters  and  various  shell  fish  have 
been  known  to  carry  the  microbic  agents  of  typhoid  fever  and  Asiatic 
cholera.  Three  infections  coming  from  meat  and  certain  other  foods 
sometimes  occur,  namely,  botulism,  enteritis  and  occasionally  para- 
typhoid fever.  In  these  instances  the  causal  microorganisms  are  in  the 
meat.  Another  type  of  infection  known  as  ptomain  poisoning  also 
occurs  from  the  eating  of  meat  or  fish  which  has  been  acted  upon  by 
saprophytic  bacteria  and  the  proteins  split  up  into  toxic  substances. 

Animal  Carriers  of  Infection. — Animals  may  communicate  disease 
microorganisms  to  one  another  and  to  man  in  three  ways,  namely, 
first,  by  direct  or  indirect  contact;  second,  by  serving  as  mechanical 
carriers  from  one  individual  to  another;  and  third,  by  serving  as  inter- 
mediate hosts  for  the  microbic  agent  and  then  subsequently  com- 
municating it  to  another.  As  examples  of  the  first  proposition,  the 
fact  that  tuberculosis  has  been  communicated  from  cattle  to  man,  that 
glanders  has  been  communicated  from  horses  to  man,  and  that 
anthrax  has  been  communicated  from  sheep  to  man  by  contact  may 
be  mentioned.  It  has  also  been  stated  that  the  cat,  while  not  suffering 
from  true  diphtheria,  seems  to  be  able  to  transmit  this  infection  and 
the  dog  may  also  transmit  rabies  to  the  man.  In  the  second  method 
of  transfer,  the  mechanical  carrying  of  an  infection,  the  insects  are 
principally  concerned.  It  is  well  demonstrated  that  common  flies 
frequently  carry  B.  typhosus  on  their  feet  from  the  infected  patient  or 
the  excreta  and  deposit  them  on  the  food  materials  thus  causing 
infection  when  the  food  is  eaten.  The  various  suctorial  insects  also 
may  suck  up  the  blood  -of  one  individual  and  carry  the  infectious 
agent  to  the  normal  individual.  Notable  examples  of  this  are  found 
in  the  transmission  of  the  various  trypanosomiases  by  the  tsetse  and 
other  tropical  flies,  and  of  Rocky  Mountain  spotted  fever  by  the  wood 
tick.  The  same  is  true  of  Bad.  pestis  of  plague  which  is  carried  by  the 
flea,  of  Texas  fever  by  the  cattle  tick,  and  it  has  been  shown  recently 
that  the  louse  may  be  one  of  the  agents  in  the  transmission  of  typhus 
fever.  As  an  example  of  the  third  method,  the  serving  as  an  inter- 
mediate host  and  the  carrying  of  the  causal  agent,  the  mosquitoes 
which  serve  as  the  only  means  of  transmission  of  the  causal  micro- 
organisms of  malaria  and  yellow  fever  and  in  which  these  parasites 
pass  a  certain  cycle  of  their  existence,  may  be  mentioned. 


668   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

Human  Carriers  of  Infection. — It  has  been  mentioned  previously 
that  man  is  capable  of  carrying  infectious  agents  when  he  himself  is 
not  infected.  For  example,  in  the  case  of  diphtheria  it  has  been 
repeatedly  shown  that  convalescents  from  diphtheria,  persons  who 
have  had  the  disease,  and  persons  who  have  never  had  the  disease, 
frequently  carry  the  etiological  microorganisms  of  this  disease  in  a 
virulent  form  and  are  accordingly  exceedingly  dangerous  as  dis- 
seminators. Not  uncommonly  persons  who  have  had  typhoid  fever 
carry  large  numbers  of  virulent  B.  typhosus  in  their  bodies,  particularly 
in  the  gall-bladder,  and  disseminate  them  through  the  intestinal 
excreta  thus  causing  many  infections  when  this  excreta  comes  into  con- 
tact with  water  used  for  drinking  purposes  or  food  supplies.  B. 
typhosus  may  be  carried  for  many  years.  Asiatic  cholera  is  occasionally 
carried  in  the  same  way.  It  has  also  been  shown  that  well  individuals 
may  carry  the  etiological  agents  of  epidemic  cerebrospinal  meningitis 
and  acute  poliomyelitis  or  infantile  paralysis.  Individuals  who  carry 
infectious  organisms  are  popularly  known  as  " bacilli  carriers"  and 
should  always  be  kept  under  rigid  quarantine  or  observation. 

Contact  Infection. — It  is  only  necessary  to  emphasize  certain  points 
in  addition  to  what  has  been  said  in  the  foregoing.  It  has  been  stated 
that  animals  may  communicate  an  infectious  agent  to  other  animals 
of  the  same  or  different,  but  susceptible,  species  by  direct  contact. 
Probably  the  commonest  diseases  to  be  communicated  by  animals  to 
each  other  are  tuberculosis  and  glanders.  This  is  commonly  accom- 
plished by  the  rubbing  of  the  mouths  and  noses  together  although  the 
disease  may  be  acquired  in  other  ways.  Among  the  human  race  the 
diseases  which  are  usually  communicated  by  the  contact  of  one  indi- 
vidual with  another  are  diphtheria,  scarlet  fever,  smallpox,  chicken- 
pox,  mumps,  measles,  gonorrhea,  chancroids  and  syphilis.  In  the  six 
first  mentioned  diseases  it  seems  that  the  expirations  and  possibly  in 
rare  instances  the  desquamations  of  the  skin  in  those  which  have  an 
eruption  carry  the  causal  microorganism.  The  infectious  agent  is 
inhaled  into  the  nose  or  throat.  Some  of  these  diseases  may  be  in 
rare  instances  transmitted  by  intermediate  agents,  clothing,  etc. 
(fomites).  In  the  last  three  diseases  mentioned,  which  are  known  as 
the  venereal  diseases,  an  abrasion  of  the  integument  is  a  prerequisite 
and  the  infectious  agent  must  enter  by  this  route.  Infection  is  usually 
brought  about  by  absolute  contact  of  one  individual  with  another. 


METHODS   AND    CHANNELS    OF   INFECTION  669 

In  leprosy  also  almost  direct  contact  is  necessary  for  a  transfer  of  the 
infectious  agent. 


THE  ROUTES  BY  WHICH  INFECTIOUS  MICROORGANISMS  ENTER  THE 

BODY 

Microorganisms  enter  the  body  through  either  the  external  or 
internal  surfaces.  It  has  been  shown  that  the  absolutely  healthy  and 
intact  skin  furnishes  an  efficient  barrier  to  the  entrance  of  infectious 
agents.  Pathogenic  bacteria,  for  example,  the  streptococcus  and  the 
various  varieties  of  the  staphylococci,  are  present  on  the  skin  almost 
continuously  yet  do  not  often  produce  infections.  When  there  is  an 
abrasion  of  the  skin  or  a  diseased  duct  or  hair  follicle  the  bacteria 
frequently  pass  down  into  the  skin  and  an  infection  results.  When 
the  pyogenic  bacteria,  such  as  mentioned  above,  are  vigorously  rubbed 
into  the  skin  infection  sometimes  takes  place  but  in  this  instance  also 
there  has  been  some  mechanical  mjury  to  the  skin.  Minute  unobserved 
abrasions  of  the  skin  also  serve  commonly  as  points  of  entrance 
for  the  Bad.  pestis  of  the  plague.  The  microorganisms  of  tetanus, 
anthrax,  symptomatic  anthrax  and  malignant  edema  always  enter 
the  skin  through  wounds.  Sometimes  the  infectious  agents  remain 
local  and  at  other  times  are  carried  from  the  point  of  the  original 
entrance.  This  may  take  place  in  different  lengths  of  time.  For 
example,  in  tetanus  the  bacteria  remain  localized  in  most  instances  at 
the  point  of  the  original  wound  and  their  toxin  diffuses  from  this 
point.  In  the  various  pyogenic  infections  the  bacteria  usually  remain 
localized.  However,  in  anthrax  the  bacteria  are  carried  into  the  circu- 
lation in  a  very  few  minutes  after  they  enter  the  wound.  In  the  new- 
born infection  very  frequently  enters  the  body  through  the  umbilicus 
or  navel.  Tetanus  is  one  of  the  common  diseases  acquired  in  this  way 
in  certain  localities.  Microorganisms  may  also  enter  the  skin  through 
the  wounds  made  by  insects  such  as  mosquitoes,  flies,  ticks,  and  fleas. 
The  larger  and  the  clearer  cut  the  wound  the  less  the  danger  of  infec- 
tion because  of  the  mechanical  and  bactericidal  barrier  of  the  fibrin  and 
the  bactericidal  action  of  the  blood  serum.  A  free  flow  of  blood  also 
washes  the  microorganisms  out  of  the  wound.  Crushing  wounds  are 
especially  dangerous  inasmuch  as  there  is  not  a  free  flow  of  blood  and 
also  there  is  a  good  chance  for  the  growth  of  anaerobic  bacteria  such 


670   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

as   those  of  tetanus,   malignant   edema,   symptomatic  anthrax  and 
emphysematous  gangrene. 

The  mucous  membranes  of  the  nose,  throat  and  mouth  are  quite 
resistant  to  infection.  The  epithelial  coat,  the  mechanical  action  of  the 
mucus  and  saliva  and  possibly  the  slight  bactericidal  action  of  the 
saliva  are  the  barriers.  Infections  of  the  thin  non-resistant  mucous 
membranes  of  the  new-born  do  occur  and  necrosis  sometimes  results 
(noma).  The  mucous  membrane  of  the  mouth  and  throat  is  frequently 
the  seat  of  primary  infection  when  it  is  injured.  The  actinomycotic 
fungus  usually  enters  a  lesion  in  the  mucous  membrane  made  by  straws 
and  other  substances.  The  ducts  of  the  salivary  glands  also  serve  as 
points  of  entrance  for  certain  infectious  agents.  The  tonsils  are  very 
commonly  the  seat  of  infections  especially  with  the  Strept.  pyogenes 
and  Strept.  pneumonia.  Septicemias,  as  for  example  those  occurring 
in  diphtheria,  and  especially  in  scarlet  fever,  frequently  arise  from  infec- 
tion of  the  tonsils  with  Strept.  pyogenes.  These  structures  are  also  the 
primary  point  of  invasion  in  cases  of  acute  rheumatism  and  possibly  in 
certain  cases  of  pulmonary  tuberculosis.  The  nasal  mucous  membrane 
is  undoubtedly  more  permeable  to  infectious  agents  than  that  of  the 
oral  cavity.  The  microorganisms  of  acute  epidemic  meningitis,  acute 
poliomyelitis,  measles,  leprosy  and  glanders  undoubtedly  most  fre- 
quently enter  the  body  through  lesions  in  the  membranes  of  the  nose. 
Infection  may  be  carried  into  the  nose  directly  or  pass  from  the 
conjunctiva  through  the  naso-lachrymal  duct. 

The  flora  found  in  the  eye  is  quite  extensive.  The  conjunctiva  is 
frequently  the  seat  of  primary  infections.  The  pyogenic  cocci  and 
the  M.  gonorrhoea  are  among  the  common  infecting  agents.  It  is 
possible  that  certain  points  of  infection  are  provided  by  the  conjunctiva 
being  injured'  by  dust  particles.  The  tears  are  not  bactericidal  and 
only  serve  to  mechanically  wash  the  eye.  Infections  of  the  con- 
junctiva are  frequently  very  severe.  There  is  no  doubt  also  that  other 
pathogens  are  caught  in  the  eye  and  washed  into  the  nose  where  they 
set  up  infections  or  are  carried  through  the  membranes  to  set  up  infec- 
tion elsewhere.  M.  intracellularis  var.  meningitidis  of  epidemic  menin- 
gitis is  known  to  pass  in  this  way  and  possibly  Bact.  pestis  of  the  plague 
in  certain  instances. 

Infectious  microorganisms  after  being  taken  into  the  body  through 
the  nose  or  the  mouth  may  either  pass  to  the  lungs  through  the  trachea 


METHODS    AND    CHANNELS    OF   INFECTION  671 

or  down  the  oesophagus  to  the  stomach  and  intestines.  During  the 
ordinary  inspiratory  part  of  a  respiration  it  is  probable  that  micro- 
organisms cannot  pass  directly  into  the  alveoli  of  the  lung  as  the 
tortuous  passage,  the  mucus  and  the  cilia  are  fairly  efficient  barriers. 
Bacteria  may  be  inhaled  directly  into  the  finer  bronchi  and  the  alveoli 
during  forced  inspiration  such  as  that  attendant  upon  hiccoughing  and 
sneezing.  Infections  of  this  kind  occur  in  pneumonia,  tuberculosis, 
and  influenza.  Microorganisms  frequently  lodge  on  the  membrane 
of  the  trachea  and  are  here  taken  up  by  the  leucocytes  and  carried  to 
the  lungs,  bronchial  lymph  glands,  and  occasionally  to  other  parts  of 
the  body  by  the  blood  and  lymph.  It  is  probable  that  such  a  form  of 
infection  occurs  sometimes  in  pneumonia,  tuberculosis  and  plague. 

Infectious  microorganisms  very  frequently  pass  down  to  the 
stomach  and  intestines.  The  mucous  membrane  of  the  stomach 
is  normally  .very  resistant  to  infection  due  to  the  hydrochloric  acid 
which  is  normally  present  in  the  gastric  juice  and  which  in  normal 
amount  is  distinctly  antiseptic.  It  should  be  remembered  that  in 
instances  where  the  acidity  of  the  stomach  is  lowered  that  .micro- 
organisms will  develop.  All  toxins  with  the  exception  of  that  of 
B.  botulinus  of  meat  poisoning  are  destroyed  by  the  gastric  juice. 
The  intestines  are  less  resistant  to  infection.  It  is  here  that  the 
causal  microorganisms  of  typhoid  fever,  Asiatic  cholera,  chicken 
cholera  and  dysentery  and  the  various  hemorrhagic  septicemias  find 
their  particular  affinities.  These  bacteria  enter  or  attach  themselves 
to  the  intestinal  wall  and  in  the  case  of  cholera  and  dysentery  this 
is  the  only  point  of  infection.  The  B.  typhosus  has  occasionally 
been  known  to  enter  at  other  places.  This  bacterium,  however,  com- 
monly localizes  in  the  lymphatic  patches  (Peyers)  of  the  intestine, 
and  may  enter  the  blood  from  this  point.  It  should  be  noted  that  some 
bacteria  can  pass  through  the  wall  of  the  intestine  when  it  is  seemingly 
intact.  This  point  has  been  repeatedly  demonstrated  in  the  case  of 
Bad.  tuberculosis. 

The  genital  organs  of  the  male  and  female  are  susceptible  to  in- 
fection with  microorganisms  in  certain  instances.  The  M.  gonorrhoea 
of  gonorrhoea  and  the  Treponema  pallidum  of  syphilis  find  their  usual 
portals  of  entry  in  the  genital  tract.  They  have,  however,  been  known 
to  infect  other  parts  of  the  body  as  the  mouth,  rectum,  and  the  con- 
junctiva. The  etiological  bacteria  of  gonorrhoea  can  penetrate  the 


672    MICROBIOLOGY  OF  DISEASES   OF. MAN  AND  DOMESTIC  ANIMALS 

seemingly  intact  male  urethra  but  not  the  vagina  of  the  female  on 
account  of  the  bactericidal  secretion  and  the  character  of  its  squamous 
epithelium.  Other  bacteria,  as  for  example,  the  Strept.  pyogenes,  M. 
pyogenes  var.  aureus  and  B.  coli  are  sometimes  found  infecting  the 
genital  tract  in  cases  of  chronic  urethritis. 

The  kidneys,  ureters  and  bladder  are  sometimes  infected  but  usually 
the  infecting  agent  is  carried  in  the  circulation  although  it  occasionally 
ascends  through  the  urethra  from  without. 

In  conclusion,  the  proposition  of  germinal  and  antenatal  infection 
must  be  mentioned.  By  true  germinal  infection  is  meant  the  carrying 
of  the  infectious  organisms  of  a  disease  by  the  ovum  or  the  spermatozoa 
and  its  incorporation  in  the  development  of  the  embryo  and  fetus. 
It  is  doubtful  if  this  ever  occurs.  Some  authorities  claim  that  it  is 
possible  and  that  it  has  been  demonstrated  that  the  spermatozoa  may 
carry  the  Treponema  pallidum  of  syphilis,  but  this  is  not  generally 
accepted.  Antenatal  infection  or  infection  of  the  fetus  before  birth 
does  occur.  Infectious  organisms  enter  the  fetus  only  as  a  result  of 
intimate  contact  with  the  mother  and  it  has  been  repeatedly  shown 
that  tuberculosis  and  syphilis  may  be  acquired  in  this  way.  It  is 
essential,  however,  that  the  mother  be  infected  and  in  most  instances 
this  infection  is  localized  in  the  placenta.  Smallpox,  scarlet  fever, 
measles,  dysentery,  various  pyogenic  infections,  and  in  rare  instances 
pneumonia,  have  also  been  acquired  by  placental  infection.  In  rare 
cases  in  animals,  anthrax,  symptomatic  anthrax,  chicken  cholera,  and 
glanders  have  been  acquired  by  antenatal  infection  from  the  mother. 

VARIATION  IN  INFECTION 

It  should  be  noted  that  there  may  be  a  variation  in  the  infection 
depending  upon  the  route  by  which  the  infectious  microorganism  enters 
the  body.  For  example,  in  the  case  of  Bad.  tuberculosis,  if  the  bacterium 
enters  the  skin  a  usually  non-fatal  infection  called  lupus  vulgaris 
results;  if  it  enters  the  lymph  glands  or  joints  and  localizes  there  an 
inflammation  of  not  necessarily  a  fatal  character  results;  if  it  enters 
the  lungs  pulmonary  tuberculosis  or  consumption  occurs  which  usu- 
ally, after  being  well  established,  runs  a  fatal  course;  if  it  enters  the 
intestine,  intestinal  tuberculosis  may  result  which  is  nearly  always 
fatal;  and  if  it  enters  the  meninges,  tubercular  meningitis  results  which 


METHODS    AND    CHANNELS    OF    INFECTION  673 

is  rapidly  fatal.  Just  so  with  the  Strept.  pyogenes,  depending  on 
whether  it  enters  the  circulation,  the  lymphatic  vessels  of  the  skin  or 
the  connective  tissues  there  results  septicemia,  erysipelas,  or  abscesses 
which  obviously  differ  in  their  severity.  The  same  is  true  of  practically 
all  the  pathogenic  bacteria  which  invade  the  plant  and  animal  body, 
the  variation  in  the  route  produces  a  great  variation  in  the  type  and 
the  results  of  the  infection. 

It  was  mentioned  in  the  beginning  of  this  discussion  that  infection 
included  certain  things  such  as  the  entrance  of  bacteria  into  the  body 
tissues,  their  increase  and  their  injury  to  the  body.  There  is  some 
variation  in  what  constitutes  an  infection  depending  upon  the  infectious 
microorganism  and  the  tissue  it  attacks.  For  example,  Msp.  comma 
of  Asiatic  cholera  does  not  produce  an  infection  unless  it  comes  into 
contact  with  the  intestinal  mucosa  and  in  this  case  it  does  not  enter 
the  tissues  but  sets  up  an  inflammatory  process  on  the  surface.  If 
this  same  bacterium  comes  into  contact  with  tissues  such  as  those  of 
the  nose,  throat,  lungs,  no  infection  results.  In  the  case  of  B.  typhpsus 
of  typhoid  fever  the  bacterium  not  only  attacks  the  intestinal  mucosa, 
but  in  addition  it  enters  the  tissue  of  the  lymphatic  patches  and  sets 
up  an  inflammation.  This  microorganism  may  also  invade  the 
circulatory  system  directly.  In  order  for  such  an  organism  as  the 
Strept.  pneumonia  to  produce  pneumonia  it  is  only  necessary  for  the 
bacteria  to  come  into  contact  with  the  thin,  single-celled,  alveolar  wall 
through  the  blood  or  air  passages.  In  case  this  bacterium  produces  an 
abscess  it  is  necessary  for  it  to  first  enter  into  the  tissues.  In  the 
pneumonic  form  of  plague,  although  infection  is  supposed  to  be  ac- 
complished generally  by  the  inhalation  of  bacilli,  the  Bact.  pestis  may 
be  carried  to  the  alveolus  through  the  circulation  and  thus  enters  the 
tissues  of  the  lung  before  actually  invading  the  alveoli.  This  some- 
times occurs  in  case  of  Strept.  pneumonia.  It  also  gives  rise  to  abscesses 
occasionally  but  only  when  it  invades  lymphatic  glands.  The  same  is 
true  of  the  large  number  of  infectious  microbic  agents;  there  is  a  varia- 
tion in  the  infection  due  to  the  variation  in  the  microorganism  and 
the  point  where  this  agent  attacks  the  body.  The  severity  of  an 
infection,  as  for  example,  a  pneumonia  due  to  Strept.  pneumonia  or 
to  Strept.  pyo genes,  or  to  Bact.  pneumonia  (Friedlander)  or  to  Bact. 
pestis,  would  vary  with  the  infectious  agent,  its  virulence  and  number, 
and  with  the  resistance  of  the  individual  infected. 

43 


674  MICROBIOLOGY  OF  DISEASES  OF  MAN  AND  DOMESTIC  ANIMALS 

THE  FACTORS  WHICH  INFLUENCE  THE  RESULTS  OF  AN  INFECTION 

There  are  four  principal  factors  which  influence  the  results  of  an 
infection.  They  are  as  follows:  The  virulence  of  the  infecting  micro- 
organism; the  number  of  the  infecting  microorganisms;  the  avenue  by 
which  the  infectious  microorganism  enters  the  body;  and  the  resistance 
of  the  plant,  animal  or  individual  infected. 

VIRULENCE. — It  is  a  self-evident  fact  that  the  more  virulent  a  micro- 
organism is  the  more  serious  will  be  the  infection  which  results  from  its 
invasion  of  the  body.  There  is  a  great  difference  in  the  virulence.  For 
example,  Strept.  pyogenes  may  infect  the  skin  of  mucous  membranes 
and  produce  only  an  abscess  of  varying  proportions.  Again,  it  may  be 
more  virulent.  The  resistance  of  the  infected  individual  may  be 
lowered  somewhat  and  the  streptococcus  may  enter  the  lymphatics 
of  the  skin  and  produce  erysipelas  or  the  blood  stream  and  produce 
a  fatal  septicemia.  Furthermore,  one  strain  of  the  streptococci  in 
the  blood  may  produce  a  very  virulent  infection  and  another  a  less 
severe  one.  Virulent  streptococci  are  not  readily  phagocytized  by  the 
leucocytes.  The  same  variation  in  virulence  is  noted  in  all  the  patho- 
genic bacteria  and  the  infections  are  modified  thereby.  The  fact  that 
an  organism  is  virulent  for  an  animal  is  not  evidence  that  it  is  virulent 
for  man.  The  virulence  of  an  organism  depends  upon  its  ability  to 
form  toxins  or  other  poisonous  substances. 

NUMBER. — The  number  of  infecting  microorganisms  which  are 
introduced  into  an  animal  body  is  of  importance.  In  anthrax,  for 
example,  it  has  been  shown  that  one  bacterium  is  capable  of  multiply- 
ing and  setting  up  an. infection.  In  tuberculosis  and  typhoid  fever  and 
most  of  the  infectious  diseases  it  requires  a  rather  large  number  before 
an  infection  will  take  place.  The  leucocytes,  bactericidal  substances 
in  the  blood,  and  the  body  cells  in  general  are  capable  of  destroying  many 
infectious  agents.  Furthermore,  it  can  be  readily  understood  how  a 
few  bacteria  might  be  able  to  cause  a  mild  infection  and  an  increasing 
number  be  able  to  so  overcome  the  bodily  resistance  as  to  cause  a  more 
or  less  severe  infection. 

AVENUE. — It  has  been  pointed  out  previously  how  the  avenue  of 
infection  modifies  the  infection.  A  very  virulent  microorganism  may 
occasionally  produce  a  very  mild  infection  when  introduced  in  a 
certain  locality  while  in  another  place  the  same  organism  may  produce 


METHODS   AND    CHANNELS    OF   INFECTION  675 

a  very  severe  type.  The  results  of  the  infection  will  be  materially 
modified  depending  on  the  avenue  of  entrance  which  the  virulent  micro- 
organism takes.  For  example,  in  addition  to  those  mentioned  previ- 
ously, suppose  Bact.  pestis,  the  causal  agent  of  plague,  enters  the 
blood  through  the  skin,  or  the  lymphatics  through  the  tonsils,  it  is 
carried  to  the  lungs  and  there  produces  a  very  severe  and  usually  fatal 
pneumonia;  if  bacteria  enter  the  lymphatic  system  in  large  numbers 
they  frequently  localize  in  the  lymph  glands  producing  buboes  or 
glandular  enlargements  which  are  not  always  fatal.  These  bacteria 
may  also  enter  the  blood  current  and  produce  a  rapidly  fatal  septicemia. 
Tt  has  not  been  established  in  man  that  plague  can  be  produced  by  the 
ingestion  of  Bact.  pestis,  but  in  some  susceptible  animals  such  as  rats, 
the  disease  in  a  very  fatal  form  is  rapidly  acquired  when  the  bacteria 
enter  the  intestines. 

RESISTANCE. — This  factor  is  one  of  the  prominent  ones  which 
modify  the  results  of  an  infection.  It  is  a  familiar  fact  that  two  or 
more  individuals  may  be  infected  with  the  same  microorganism,  as 
for  example,  B.  typJiosus,  and  one  will  not  become  infected  or  have  a 
very  mild  form  of  the  disease,  while  the  other  will  have  the  severest  and 
most  fatal  form  of  typhoid  fever  known.  Again,  the  age  of  the  indi- 
vidual infected  is  important  in  determining  the  resistance.  The  adult 
resists  infection  such  as  diphtheria,  scarlet  fever,  and  measles  more 
than  the  child.  The  very  young  child  resists  pneumonia  and  tuber- 
culosis more  than  the  adult.  The  resistance  of  the  body  depends  on 
the  presence  in  that  body  of  natural  or  acquired  antibodies.  It  is, 
therefore,  obvious  that  the  higher  resistance  or  immunity  of  the  indi- 
vidual infected,  the  less  severe  will  be  the  results  of  the  infection  on 
that  individual. 


THE    EXACT    CAUSE    OF    INFECTIONS 

We  are  familiar  with  the  fact  that  all  of  our  infectious  diseases  are 
due  to  microorganisms  or  viruses  of  some  form  or  other.  The  causal 
agents  of  many  of  these  diseases  are  known  but  in  the  case  of  those  that 
are  not  known  there  is  reasonable  certainty  as  to  the  types  of  the  in- 
fecting agents.  The  exact  substances  which  are  produced  by  the  micro- 
organisms and  which  are  responsible  for  symptoms  of  the  various 
diseases  will  be  briefly  considered  in  the  following  paragraphs. 


676   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

SOLUBLE  TOXINS. — It  is  a  known  fact  that  there  are  some  patho- 
genic bacteria  which  secrete  through  their  cell  walls  poisons  which 
diffuse  into  the  surrounding  media.  To  these  poisons  or  toxins  the 
disease  symptoms  are  due.  Bact.  diphtheria  of  diphtheria,  B.  tetani 
of  lock-jaw  or  tetanus,  Bact.  dysenteries  of  bacillary  dysentery,  B. 
botulinus  of  meat  poisoning,  and  Ps.  pyocyanea,  the  causal  organism  of 
blue-green  pus,  are  about  the  only  bacteria  of  this  character.  Some 
bacteria,  such  as  Strept.  pyogenes  and  M.  pyogenes  var.  aureus  produce 
hemolytic  toxins.  There  are  certain  protozoa  as,  for  example,  cer- 
tain entamoebse  and  the  various  trypanosomes  which  secrete  soluble 
poisons.  Among  the  animals,  the  venoms  of  the  poisonous  snakes,  the 
poison  of  the  centipedes  and  spiders,  the  serum  of  the  eel,  and  the 
excretion  of  the  dermal  glands  of  the  toad  are  examples  of  secreted 
toxins  (zoo toxins).  Again,  among  the  plants  abrin  from  the 
jequerity  bean,  ricin  from  the  castor  oil  bean,  and  others,  are  examples 
of  soluble  toxins  the  product  of  plant  cells  (phytotoxins).  The  cells 
producing  these  toxic  substances,  therefore,  are  only  indirectly  re- 
sponsible for  the  infections  for  it  is  the  toxins  themselves  which  produce 
the  pathogenic  effect  on  the  body. 

ENDOTOXINS. — Many  of  the  pathogenic  bacteria  and  some  of  the 
protozoa  do  not  secrete  their  toxins  outside  the  cell  wall  but  hold 
them  within  the  wall  in  combination  with  the  protoplasm.  They  do 
not  liberate  these  substances  until  the  microorganisms  die  and  are 
disintegrated.  Such  toxic  substances  are  called  endotoxins  to  dis- 
tinguish them  from  those  secreted  from  the  cell,  namely,  the  soluble 
toxins.  Two  of  the  best  examples  of  pathogenic  bacteria  of  this 
type  are  the  Msp.  comma  of  Asiatic  cholera  and  B.  typhosus  of 
typhoid  fever. 

Toxic  BACTERIAL  PROTEINS. — There  are  some  bacteria  and  other 
parasitic  cells  which  produce  a  small  amount  of  endotoxin  and  in 
certain  instances  some  soluble  toxin  but  not  enough  of  either  of  these 
substances  to  account  for  the  toxicity  of  the  organism.  It  has  been 
found  that  when  organisms  of  this  character  are  ground  up  and  washed 
to  free  them  of  their  endotoxin  and  are  washed  free  of  all  soluble 
toxins,  they  are  still  toxic.  It  has  been  shown  that  this  toxicity  is  due 
to  the  protein  substances  of  the  cell.  The  Bact.  tuberculosis  and  the 
Bact.  mallei  of  glanders  are  two  notable  examples  of  microorganisms 
of  this  character.  When,  for  example,  the  proteins  of  Bact.  tuberculosis 


METHODS    AND    CHANNELS    OF    INFECTION  677 

are  injected  into  the  circulation  of  susceptible  animals,  tubercle 
formation  occurs  showing  that  these  proteins  are  poisonous. 

OTHER  POSSIBLE  EXACT  CAUSES. — In  certain  infectious  diseases  it 
is  also  claimed  by  certain  writers  that  enzymes  are  responsible.  This 
lacks  substantiation.  It  is  also  stated  that  in  such  infections  as  anthrax 
the  mechanical  effect  of  the  bacteria  plugging  up  the  capillaries  and 
producing  mycotic  emboli  is  a  factor.  This  may  be  true  but  in  addition 
other  factors  are  concerned  as  previously  mentioned. 

In  mixed  infections  of  two  or  more  organisms,  which  frequently 
occurs,  the  infected  individual  may  have  within  the  body  soluble 
toxins,  endotoxins,  and  toxic  bacterial  proteins  and  in  such  a  case  it  is 
difficult  to  differentiate  their  action. 


THE  METHODS  BY  WHICH  INFECTIOUS  MICROORGANISMS  ARE 
DISSEMINATED 

The  microorganisms  of  some  of  the  infectious  diseases  such  as  diph- 
theria and  Asiatic  cholera  and  usually  tetanus  remain  local  and  seldom 
enter  the  body  generally.  From  the  locus  of  the  infection  they  dis- 
seminate their  toxic  or  poisonous  products.  In  the  case  of  tetanus  the 
toxin  is  carried  over  the  body  along  the  sheaths  of  the  motor  nerves; 
in  diphtheria  the  toxin  is  usually  carried  by  the  lymph,  occasionally 
by  the  blood;  and  in  the  case  of  cholera  the  blood  and  lymph  both  serve 
to  carry  the  toxic  agents.  In  diphtheria  and  cholera  the  microorgan- 
isms very  frequently  extend  along  the  mucous  membranes  from  the 
original  point  of  infection.  There  are  other  infections  in  which  the 
causal  microorganisms  extend  only  from  the  point  of  original  invasion 
into  the  surrounding  areas.  Such  is  the  case  with  Strept.  pyogenes  in 
the  infection  of  the  lymphatics  of  the  skin  in  erysipelas  and  of  Bad. 
influenza  in  all  infection  through  the  respiratory  tract.  Many  infec- 
tious agents  are  carried  by  the  blood  and  occasionally  by  the  lymph, 
as  for  example,  in  tuberculosis,  syphilis,  glanders,  plague,  leprosy, 
pneumonia,  and  the  septicemias  due  to  the  pyogenic  cocci.  It  is  pos- 
sible in  certain  cases  that  the  leucocytes  acting  as  phagocytes  may 
carry  virulent  infectious  agents  through  the  blood  and  lymph  from  one 
part  of  the  body  to  the  other. 


678    MICROBIOLOGY   OF  DISEASES    OF   MAN  AND   DOMESTIC   ANIMALS 

THE  METHODS  BY  WHICH  INFECTIOUS  AGENTS  ARE  ELIMINATED  FROM 

THE  BODY 

The  etiological  microorganisms  of  the  various  infectious  diseases 
may  be  eliminated  from  the  body  in  two  general  ways,  namely,  by  a 
direct  method  and  by  an  indirect  method.  For  a  microorganism  to 
be  directly  eliminated  from  the  body  it  is  necessary  for  the  focus  of  the 
infection  to  communicate  with  the  outside  of  the  body  in  some  way  or 
other.  In  the  case  of  infections  of  the  mucous  membranes  and  the  skin 
there  is,  of  course,  direct  communication  with  the  outside.  In  diseases 
of  the  respiratory  organs  and  the  intestines  the  infectious  agents  are 
discharged  into  the  lumen  of  the  air  passages  and  the  intestines  and  then 
thrown  out  from  these  passages.  Examples  of  the  partial  direct  elimi- 
nation from  the  skin  may  be  found  in  such  diseases  as  smallpox,  measles, 
syphilis,  scarlet  fever,  lupus  vulgaris,  and  in  suppurative  conditions 
such  as  carbuncles  and  furuncles.  From  the  present  evidence  little 
significance  perhaps  is  to  be  attached  to  the  elimination  of  the  infectious 
agents  mentioned  directly  from  the  skin.  It  is  probable  that  the  micro- 
organisms which  are  eliminated  remain  alive  for  only  a  short  time  and  are 
not  factors  of  consequence  in  the  transmission  of  these  infections.  As 
examples  of  diseases  in  which  direct  elimination  from  the  various 
mucous  membranes  occurs,  infections  such  as  typhoid  fever,  tubercu- 
losis, cholera  and  dysentery  from  the  membranes  of  the  intestines; 
influenza,  pneumonia  and  tuberculosis  from  the  bronchial  membranes; 
diphtheria,  leprosy,  glanders  and  scarlet  fever  from  the  membranes  of 
the  nose,  throat,  and  tonsils;  and  gonorrhea,  syphilis  and  tuberculosis 
from  the  membranes  of  the  genito-urinary  tract  may  be  mentioned. 
In  elimination  from  the  various  internal  membranes  sometimes  re- 
infections occur  such  as  in  the  case  of  the  elimination  of  Bact.  tubercu- 
losis from  the  respiratory  tract,  the  swallowing  of  the  sputum,  and  the 
subsequent  infection  of  the  intestines. 

In  the  second,  or  indirect  method  of  elimination,  two  distinct  prop- 
ositions present  themselves;  first,  the  infectious  microorganism  must 
enter  the  lymphatic  or  blood  circulation;  and,  secondly,  in  order  to  get 
out  of  the  body  they  must  pass  through  the  cells  of  some  of  the  organs, 
the  mucous  membranes  or  skin.  It  is  a  common  occurrence  for  bac- 
teria and  other  microorganisms  to  get  into  the  circulation  in  some  of 
the  infectious  diseases  such  as  typhoid  fever,  pneumonia,  plague, 


METHODS   AND    CHANNELS    OF   INFECTION  679 

and  in  the  various  septicemias.  They  may  pass  through  the  epithelium 
of  the  kidney  and  be  eliminated  in  the  urine;  they  may  pass  through  the 
liver  and  be  eliminated  in  the  bile,  finally  passing  out  through  the  in- 
testines; and  they  may  pass  through  the  mucous  membranes  of  the 
intestine  and  possibly  pass  through  the  glandular  epithelium  of  the 
sebaceous  and  sweat  glands  and  be  eliminated  through  the  skin.  They 
have  also  been  known  to  pass  through  the  glandular  epithelium  of  the 
milk  glands  when  these  glands  are  not  grossly  diseased  and  through  the 
salivary  glands.  It  has  been  recently  well  demonstrated  that  there 
must  be  some  form  of  lesion  in  the  liver  and  kidney  in  order  for  the 
microorganisms  to  pass  through.  Infectious  microorganisms  are  some- 
times destroyed  by  the  lysins  in  the  blood,  carried  to  and  deposited 
in  the  spleen  and  bone  marrow  and  gradually  disintegrated  and 
dissolved. 

In  certain  infections  in  which  a  recovery  seems  to  have  occurred 
all  the  infectious  microorganisms  are  not  always  eliminated  from  the 
body.  As  mentioned  previously,  B.  typhosus  and  Bact.  diphtheria  are 
frequently  carried  by  persons  fully  recovered  from  these  diseases. 
Sometimes,  however,  inflammatory  infections  are  set  up  by  these  bac- 
teria. It  has  been  suggested  on  seemingly  good  evidence  that  inflam- 
mations of  the  gall-bladder  and  gall-stone  formation  may  be  due  to  the 
toxic  action  of  the  bacteria  of  typhoid  fever  which  have  been  retained 
in  the  gall-bladder  for  a  considerable  time  following  an  attack  of  typhoid 
fever.  It  is  known  that  frequently  repeated  attacks  of  malaria  are  due 
to  the  retention  of  some  of  these  protozoan  parasites  for  a  time  in  the 
quiescent  stage.  Repeated  attacks  of  erysipelas  caused  by  the  Strept. 
pyogenes  may  also  be  due  to  the  same  condition.  It  is  also  claimed  by 
some  (Von  Behring)  that  Bact.  tuberculosis  is  taken  into  the  body  in 
infancy,  that  it  is  not  eliminated,  and  that  it  sets  up  infection  in  later 
life. 

In  conclusion  should  be  mentioned  one  other  indirect  way  in  which 
infectious  agents  are  eliminated  from  the  body,  namely,  by  being 
taken  up  by  suctorial  insects  from  the  blood.  It  is  necessary  that  this 
be  done  in  order  to  perpetuate  the  parasite  and  complete  its  life  cycle  in 
certain  instances,  as  with  the  mosquitoes  in  yellow  fever  and  malaria. 
In  other  instances  the  parasites  are  only  taken  up  by  the  insect  ana 
subsequently  injected  into  another  individual  or  digested  as  the  case 
may  be.  This  occurs  with  the  ticks  in  the  transmission  of  certain  of 


68o   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND   DOMESTIC  ANIMALS 

the  piroplasmoses  and  with  the  tsetse  flies  in  the  transmission  of 
certain  of  the  trypanosomiases. 

THE  EFFECT  OF  INFECTIOUS  MICROORGANISMS  ON /THE  BODY 

It  becomes  necessary  to  consider  briefly  the  effect  of  the  various 
infectious  microorganisms  and  their  toxic  substances  on  the  body. 

THE  PERIOD  OF  INCUBATION. — This  period  is  that  elapsing  after 
the  entrance  of  the  infecting  organism  into  the  body  until  the  symp- 
toms of  the  disease  develop.  .  This  period  is  variable  in  most  diseases 
and  depends  upon  the  same  factors  which  modify  the  results  of  an 
infection,  namely,  the  virulence  of  the  infecting  organism,  the  number 
growing  in  the  body  or  its  tissues,  the  avenue,  and  the  resistance  of  the 
individual.  The  period  can  be  in  a  measure  controlled  and  shortened 
in  experimental  animals  by  inoculations  into  the  circulatory  system 
and  in  other  regions  depending  on  the  organisms  used.  In  some  of 
the  human  diseases,  particularly  those  of  unknown  cause,  the  period  of 
incubation  is  quite  constant,  as  for  example,  in  smallpox  and  measles. 

LOCAL  REACTIONS. — The  local  effects  of  the  toxic  substances  of 
microorganisms  are  usually  first  inflammatory  and  later  possibly  ne- 
crotic,  that  is,  they  produce  a  death  of  the  tissue  involved.  The  inflam- 
matory changes  may  be  confined  to  those  of  an  acute  character  as,  for 
example,  in  the  various  serous,  hemorrhagic,  suppurative  and  fibrinous 
inflammations,  or  be  chronic  and  proliferative  in  nature.  There  is 
always  a  variation  in  the  type  of  inflammation  depending  on  the  loca- 
tion of  the  infection  and  also  a  variation  in  two  different  individuals  of 
the  same  species  infected  at  the  same  point  with  the  same  agent.  In 
some  diseases  such  as  tetanus  the  local  point  of  infection  may  entirely 
heal  and  still  the  bacteria  be  localized  at  this  point  and  disseminate 
their  toxin.  In  some  cases  of  tuberculosis  and  glanders  the  bacteria 
may  become  localized  at  the  points  of  infection  and  after  an  acute 
inflammatory  stage  the  point  may  become  the  seat  of  a  chronic  process 
and  proliferative  changes  occur  in  the  tissues. 

GENERAL  REACTIONS. — Metabolism. — The  general  metabolism  of 
the  body  is  affected  by  the  changes  produced  in  the  amount  and  the 
chemical  constitution  of  the  food  substances  which  are  taken  into 
the  body.  By  changing  in  the  same  way  the  substances  which  natu- 
rally are  eliminated  from  the  body  and  by  setting  up  new  and  abnormal 


METHODS    AND    CHANNELS    OF    INFECTION  68 1 

changes  in  the  functional  activity  of  the  tissues,  the  general  metabolism 
may  be  disturbed.  Muscular  weakness,  delirium,  pain  and  loss  of 
appetite,  together  with  vomiting,  diarrhea,  disturbance  of  intestinal 
absorption  and  the  digestive  juices  are  often  noted  in  cases  of  altered 
metabolism.  The  fats,  carbohydrates,  and  then  the  proteins  are  in 
the  order  named  rapidly  used  up,  producing  certain  changes  in  the 
respired  air  and  in  the  urine  and  feces.  Infectious  microorganisms 
may  also  reduce  the  power  of  the  hemoglobin  to  carry  oxygen  and 
perhaps  cause  a  narrowing  of  the  respiratory  passages  thus  preventing 
the  necessary  amount  of  oxygen  reaching  the  lungs  and  subsequently 
the  tissues. 

Infecting  microorganisms  may  alter  the  composition  of  the  food 
substances  and  after  being  taken  into  the  body  produce  abnormal 
compounds  which  have  little  or  no  nutritive  value  on  absorption  or 
they  may  produce  toxic  substances  related  to  ptomains.  Substances 
which  normally  should  be  eliminated  from  the  body  are  often  retained 
and  abnormal  losses  of  such  substances,  as  water  and  various  albumi- 
nous compounds,  occur. 

Attendant  upon  the  changes  in  metabolism  usually  there  occurs 
fever  in  all  infectious  diseases.  It  is  probable  that  the  fever  is  the 
result  of  the  effect  of  the  toxic  protein  compounds  of  the  infecting 
microorganisms  on  the  tissues,  or  the  disintegration  of  the  protein 
compounds  of  the  body  due  to  the  action  of  toxins.  It  is  evident  that 
the  fever-producing  substances,  in  certain  infectious  diseases,  act  in  a 
very  characteristic  manner  as  is  demonstrated  by  the  so-called  typical 
fever  curves.  It  seems  to  have  been  demonstrated  that  fever  is  a  good 
sign  and  that  it  is  indicative  of  the  reaction  of  the  body  to  the  toxic 
substances  of  the  infecting  agents.  It  has  also  been  shown  that  the 
fall  of  fever  in  certain  infections  is  attendant  upon  the  formation  and 
saturation  of  the  body  fluids  with  antibodies. 

Blood-forming  Organs. — There  are  usually  changes  in  the  blood- 
forming  organs  in  most  all  types  of  infection.  The  spleen  frequently 
shows  enlargement  and  contains  numbers  of  myelocytes  which  are 
also  found  in  the  blood  in  increased  numbers.  This  is  probably  due  to 
the  disintegration  and  deposition  there  of  red  blood  corpuscles  and 
also  to  the  action  of  toxic  substances.  The  endothelial  cells  and 
leucocytes  of  the  spleen  are  actively  phagocytic.  The  bone  marrow, 
particularly  the  fatty  marrow,  shows  large  numbers  of  myelocytes  of 


682    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

the  neutrophile  type  and  it  eventually  becomes  lymphoid  in  nature 
in  a  large  number  of  infections.  The  lymph  glands  also  frequently 
show  endothelial  proliferation. 

Parenchymatous  Tissues. — All  sorts  of  degenerations  of  the  kidneys, 
heart,  liver  and  some  of  the  other  organs  frequently  occur  in  infections. 
Amyloid  formation  and  necrosis  of  tissue  sometimes  occurs.  Many  of 
the  toxins  have  special  affinities  for  tissues,  such  as  the  tetanus  toxin 
for  nerve  tissue  and  this  produces  organic  changes.  It  is  possible 
also  that  the  fever  is  responsible  for  a  certain  portion  of  the  changes 
in  the  parenchymatous  organs  in  infections. 

Epithelial  and  Endothelial  Tissues, — In  certain  infections,  as  for 
example,  diphtheria,  the  epithelial  tissues  are  subject  to  inflammation; 
and  in  other  infections,  as  for  example,  syphilis,  the  endothelial  tissues 
of  the  blood-vessels  undergo  inflammation  and  sometimes  proliferation. 
The  epithelial  and  endothelial  cells  are  frequently  actively  phagocytic 
and  large  numbers  of  the  infecting  microorganisms  are  taken  up  and 
destroyed.  Some  of  the  infectious  microorganisms  produce  no  effect 
whatever  on  these  tissues,  while  others  produce  pronounced  destructive 
changes. 

Erythrocytes  and  Leucocytes.— Lytic  or  dissolving  substances  for 
the  red  blood  corpuscles  are  frequently  produced  in  infections  (hemo- 
lysins).  Strept.  pyogenes,  M.  pyogenes  var.  aureus,  and  Ps.  pyocyanea 
are  among  the  bacteria  which  produce  hemolysins.  Anemia  is,  therefore, 
not  an  uncommon  finding  in  many  infections.  Normal  human  blood 
and  that  of  some  animals  contains  an  antilysin  for  the  staphylolysin  and 
it  is  sometimes  produced  in  large  amounts.  Agglutinating  sub- 
stances for  red  corpuscles  are  produced  by  some  pathogenic  micro- 
organisms and  it  is  possible  that  these  are  the  cause  of  the  so-called 
agglutination  thrombi  which  occur  in  infections  like  typhoid  fever. 

The  most  marked  changes  seen  in  the  leucocytes  in  infections  is 
their  rather  constant  increase  in  number;  in  most  cases  a  leucocytosis. 
In  uncomplicated  tuberculosis  and  typhoid  fever,  in  measles  and 
German  measles,  in  malaria,  and  in  dengue,  there  is  no  increase  in 
number.  In  acute  inflammations  it  is  the  polymorphonuclear  leuco- 
cytes that  undergo  an  increase.  This  increase  is  sometimes  preceded 
by  a  decrease  (leucopenia).  The  leucocytes  act  as  the  principal  phago- 
cytes of  the  body  and  are  attracted  (positive  chemotaxis)  to  the  bacteria 
or  other  microorganisms  after  they  have  been  sensitized  by  theopsonins 


METHODS    AND    CHANNELS    OF    INFECTION  683 

in  the  body  fluids.  Besides  acting  as  phagocytes  they  may,  according 
to  Metchnikoff,  produce  antitoxins  and  bactericidal  substances.  It 
has  been  suggested  that  the  initial  leucopenia  in  some  cases  is  due  to  the 
production  of  negative  chemotactic  substances.  Some  virulent 
bacteria  cannot  be  phagocytized  probably  because  they  produce 
very  strong  negative  chemotactic  substances. 

Antibody  Formation. — One  of  the  general  results  of  pathogenic  micro- 
organisms in  certain  infections  is  the  production  of  antibodies  of  various 
kinds.  These  may  be  antitoxins  as  in  the  case  of  tetanus  and  diph- 
theria, or  bactericidal  substances  as  in  typhoid  fever  and  cholera,  or 
opsonic  substances  as  in  the  pyogenic  infections.  Agglutinins,  pre- 
cipitins,  and  other  bodies  are  also  sometimes  produced  in  conjunction 
with  the  other  immune  substances  mentioned. 


CHAPTER  II* 
IMMUNITY  AND  SUSCEPTIBILITY 

GENERAL 

DEFINITION. — A  clear  understanding  as  to  what  is  meant  by  the 
terms  immunity  and  susceptibility  is  of  fundamental  importance. 
By  immunity  we  mean  the  resistance  which  ah  animal  or  plant  body 
possesses  to  the  etiological  microorganisms  of  an  infectious  disease  and 
to  the  disease  itself.  The  name  has  been  adapted  from  the  Latin 
immunis  which  meant  a  person  who  was  free  or  exempt  from  public 
duties  and  later,  one  who  was  exempt  from  the  action  of  poisons. 
Briefly  stated,  immunity  is  resistance  to  disease.  It  results  commonly  as 
a  natural  termination  of  the  process  of  self-healing  in  many  infectious 
diseases.  The  absence  of  such  resistance,  which  may  be  total  or  partial, 
characterizes  what  is  known  as  susceptibility.  Throughout  the  animal 
kingdom  and  also  among  the  plants  there  is  a  great  variation  in  the 
immunity  and  susceptibility  in  the  different  species  to  the  various  dis- 
eases. Immunity  bears  no  relation  to  the  contagiousness  of  a  disease 
and  the  term  is  only  applied  as  a  rule  to  strictly  infectious  diseases  and 
not  to  metabolic  diseases. 

HYPERSUSCEPTIBILITY  OR  ANAPHYLAXIS. — It  has  been  shown  that 
animals  and  man  are  occasionally  hypersusceptible  to  certain  proteins. 
For  example,  there  are  individuals  who  are  always  seriously  poisoned 
by  the  ingestion  of  eggs,  pineapples,  and  strawberries.  Certain  indi- 
viduals when  injected  with  diphtheria  or  tetanus  antitoxin  which  is 
carried  in  horse  serum  are  seriously  intoxicated  and  occasionally  die. 
In  such  instances  the  proteins  of  the  horse  serum  and  not  the  antitoxin 
are  responsible.  It  has  been  demonstrated  that  an  animal  may  be 
sensitized  or  rendered  hypersusceptible  to  almost  any  protein  by  first 
injecting  a  minute  amount  of  the  protein  and  then,  after  a  period  of  at. 
least  eight  to  thirteen  days,  may  be  seriously  intoxicated,  if  not  killed, 
by  the  injection  of  a  slightly  increasing  dose  of  the  same  protein.  The 

*  Prepared  by  E.  F.  McCampbell. 

684 


IMMUNITY    AND    SUSCEPTIBILITY  685 

proteins  of  the  bacterial  cells  have  been  shown  to  act  in  the  same  way. 
Animals  injected,  as  described  above,  may  be  rendered  hypersusceptible 
to  all  bacterial  proteins.  Furthermore,  as  referred  to  above,  individuals 
may  be  naturally  hypersusceptible  to  bacterial  and  other  proteins. 
The  manner  of  the  original  sensitization  in  these  cases  is  not  known. 
Sensitization  which  has  been  either  naturally  or  artificially  acquired 
is  transferred  in  most  instances  in  utero  to  the  first  generation;  that  is, 
a  mother  may  be  sensitized,  convey  the  sensitizing  substances  to  her 
young  while  in  the  uterus,  and  when  these  offsprings  are  subsequently 
injected  after  birth  with  the  same  protein  they  may  be  intoxicated  or 
killed.  In  this  connection  it  should  be  stated  that  the  so-called  in- 
herited tendency  to  specific  diseases  may  be  something  more  definite 
than  we  are  ordinarily  accustomed  to  regard  it.  Suppose  a  mother 
becomes  tuberculous  and  is,  therefore,  sensitized  to  the  proteins  of  Bact. 
tuberculosis;  it  is  quite  possible  for  her  to  convey  to  the  offspring  this 
susceptibility  to  the  particular  proteins  of  Bact.  tuberculosis  as  has  been 
demonstrated  artificially.  After  birth,  or  in  later  life,  when  the  causal 
microorganisms  of  tuberculosis  are  taken  into  the  body,  as  they  are  in 
about  ninety-five  per  cent  of  all  persons  in  civilized  countries,  the 
bacteria  may  find  a  more  than  ordinarily  susceptible  individual  and 
may  develop  with  comparatively  little  hindrance.  If  this  condition 
be  true  naturally  as  it  is  when  produced  experimentally  in  suscep- 
tible animals  a  very  interesting  and  scientific  explanation  of  the 
so-called  inherited  tendency  to  tuberculosis  is  at  hand. 

PREDISPOSITION  AND  NON-INHERITANCE  OF  INFECTIOUS  DISEASES. 
There  is  probably  no  such  thing  as  a  truly  inherited  infectious  disease. 
This  point  has  been  debated  and  discussed  for  a  great  many  years  and 
the  above  conclusion  has  been  reached  by  the  majority  of  investigators. 
By  inheritance  is  meant  the  transference  of  a  property,  or  in  this  in- 
stance, a  pathogenic  microorganism  by  the  nuclear  substance  of  either 
the  spermatozoon  or  the  ovum.  It  is  only  the  nuclear  substances  which 
combine  to  form  the  new  individual.  It  is  true  among  certain  of  the 
lower  animals,  such  as  the  fowls  and  some  insects,  that  microorganisms 
are  carried  within  the  egg  but  the  eggs  are  quite  different  in  structure 
from  the  human  or  mammalian  ovum.  The  egg  of  the  above-mentioned 
animals  is  composed  largely  of  yolk-furnishing  food  and  there  is  ample 
opportunity  for  microbic  growth,  while  the  mammalian  ovum  contains 
no  yolk.  Such  instances  should  be  referred  to  as  germ-cell  transmission, 


686   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

not  inheritance.  If  the  microorganisms  were  present  they  would  be 
immediately  incorporated  within  the  new  developing  embryo.  If  the 
microorganism  ever  did  find  its  way  into  the  human  or  mammalian 
germ  cells  it  would  be  a  mechanical  impossibility  for  the  cells  of  the 
embryo  to  divide  and  multiply  in  proper  manner.  Such  pathogens 
would  rapidly  destroy  the  developing  cells  in  the  embryo.  It  is  true 
that  the  offspring  of  certain  individuals  are  born  diseased.  For  ex- 
ample, children  are  not  infrequently  born  with  syphilis  and  tuberculosis. 
At  first  thought  it  might  seem  that  this  is  inheritance  but  on  careful 
analysis  it  will  be  found  that  the  mother  is  either  syphilitic  or  tubercu- 
lous. Furthermore,  the  focus  of  the  infection  is  most  frequently  in  the 
uterus  and  the  microorganisms  are  transferred  to  the  unborn  offspring 
by  means  of  the  fetal  circulation.  This  condition  is  what  is  known  as 
antenatal  acquirement;  it  is  not  heredity.  It  is  absolutely  impossible  for 
the  male  to  communicate  any  disease  to  the  offspring  unless  the  female 
is  first  infected.  Colle  years  ago  formulated  a  law  which  bears  his  name 
in  which  he  stated  that  a  father  could  communicate  syphilis  to  his  child 
without  the  mother  being  infected.  This  law  has  been  disproved  since 
the  introduction  of  the  new  serum  tests  for  syphilis  and  it  can  be  posi- 
tively demonstrated  in  all  such  cases  that  the  mother  is  infected.  Ante- 
or  prenatal  acquirement  may  then  be  recognized.  What  can  be  said 
in  regard  to  the  predisposition  to  a  definite  infectious  disease?  There 
is  a  question  as  to  whether  true  predisposition  does  exist.  Many 
cases  are  on  record  to  show  that  disease  seems  to  run  in  families  and  in 
localities.  For  example,  tuberculosis  and  cancer  are  frequently  said 
to  be  subject  to  inheritance  or  to  predisposition  in  certain  cases.  It 
can  be  easily  seen  that  if  one  parent  is  diseased  the  germ  cell  of  the 
parent  will  be  less  healthy  and  when  combined  with  a  normal  healthy 
germ  cell  of  the  other  parent  will  not  give  rise  to  as  healthy  an  individual 
as  when  both  cells  are  from  healthy  individuals.  Again,  the  result  when 
the  germ  cells  of  both  parents  are  unhealthy,  due  to  the  parents  being 
unhealthy,  is  evident.  Predisposition  seems  to  resolve  itself  into  the 
inheritance  of  a  weakened  constitution,  a  constitution  which  will  not 
withstand  the  ordinary  infections  easily.  It  seems  not  to  be  a  predis- 
position to  any  particular  disease  but  a  predisposition  to  all  diseases, 
infectious  and  metabolic.  Diseases  such  as  tuberculosis  are  so 
prevalent  that  it  is  very  possible  that  infection  may  take  place  and  it  be 
interpreted  as  inherited  because  the  parent  died  of  the  same  cause. 


IMMUNITY  AND    SUSCEPTIBILITY  687 

As  mentioned  above,  it  may  be  that  the  true  explanation  of  the 
phenomena  of  predisposition  is  found  in  anaphylaxis  or  the  sensitiza- 
tion  to  various  proteins  of  microorganisms.  Further  work  is  necessary 
along  these  lines. 

IMMUNITY 

Immunity  and  susceptibility  to  disease  are  always  relative  and 
never  absolute;  that  is,  it  is  always  possible  to  produce  some  sort  of  an 
infection  in  a  supposedly  immune  animal  by  modifying  the  conditions 
under  which  the  animal  is  accustomed  to  live.  For  example,  the 
chicken  is  immune  to  tetanus  but  by  keeping  this  animal  for  some  time 
at  a  temperature  higher  than  its  normal  it  may  be  infected.  The  cow 
cannot  ordinarily  be  infected  with  typhoid  but  when  large  numbers  of 
the  B.  typhosus  are  injected  under  the  skin  an  abscess  may  be  produced. 
These  and  many  other  examples  might  be  mentioned.  Our  standard 
of  immunity  in  a  particular  animal  is  based  upon  the  conditions  as  they 
exist  naturally  and  on  the  average  resistance  of  animals  of  the  same 
species. 

Immunity  to  disease  may  be  of  two  kinds,  natural  and  acquired. 
Natural  immunity  is  that  resistance  which  is  possessed  normally  by  an 
individual.  Acquired  immunity  is  that  resistance  which  is  acquired 
by  having  an  infection,  or  by  being  vaccinated,  or  immunized  against 
an  infection  with  the  specific  etiological  microorganism  or  its  antiserum. 

NATURAL  IMMUNITY  AND  SUSCEPTIBILITY. — Attention  should  be 
directed  to  certain  forms  of  natural  immunity  and  susceptibility. 

Racial  Immunity  and  Susceptibility. — It  is  a  familiar  fact  that  certain 
species  of  animals  and  certain  races  of  man  differ  in  their  resistance  and 
their  susceptibility  to  infectious  diseases.  As  examples  of  racial  im- 
munity among  animals  the  native  cattle  of  Austria  and  Hungary  and  of 
Japan  which  are  relatively  immune  to  bovine  tuberculosis,  a  disease 
which  causes  great  loss  among  other  breeds,  may  be  mentioned.  Again, 
the  sheep  of  Algeria  are  relatively  immune  to  anthrax  while  all  other 
sheep  are  extremely  susceptible.  Field  mice  are  immune  to  glanders 
while  the  common  house  mouse  is  susceptible.  The  negro  is  more  re- 
sistant to  the  infectious  microbic  agent  of  yellow  fever  than  other  races, 
but  is  without  doubt  more  susceptible  to  tuberculosis.  The  Japanese 
are  said  to  be  more  resistant  to  scarlet  fever  than  other  races.  The 


688   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

Melanesians  are  very  susceptible  to  measles  and  the  Malaysians  to  beri- 
beri, while  other  races  are  relatively  immune. 

Familial  Immunity  and  Susceptibility. — It  is  true  that  certain 
families  vary  in  their  immunity  and  susceptibility  when  compared  with 
other  families  in  the  same  community.  For  example,  tuberculosis 
undoubtedly  shows  a  tendency  to  run  in  families.  In  determining  a 
case  of  this  kind  it  is,  of  course,  necessary  to  take  cognizance  of  the 
environment  of  the  individual  and  the  association  with  other  diseased 
persons.  The  so-called  tuberculous  diathesis  does  exist  and  perhaps  we 
have  an  explanation  of  it  in  anaphylactic  phenomena  as  mentioned 
previously.  Measles  and  scarlet  fever  also  in  certain  instances  seem  to 
run  in  families. 

Individual  Immunity  and  Susceptibility. — Variation  among  indi- 
viduals associated  together  is  noted  in  regard  to  their  resistance  and 
susceptibility  to  disease.  It  is  well  known,  for  example,  that  in  a  herd 
of  cattle,  which  are  in  the  main  tuberculous,  there  are  certain  individuals 
who  never  contract  the  disease.  These  animals  may  be  of  the  same 
breed  and  be  fed  and  handled  the  same  as  the  rest  of  the  herd,  still  they 
never  become  infected.  Again,  in  the  human  race,  with  the  acute  exan- 
thematous  diseases  such  as  scarlet  fever  and  measles,  there  are  children, 
for  example,  in  the  same  family  and  of  nearly  the  same  age  and  living 
under  exactly  the  same  conditions,  who  contract  the  disease  and  others 
who  do  not.  The  exact  cause  of  the  individual,  familial  and  racial 
immunity  cannot  be  satisfactorily  explained  at  the  present  time.  There 
is  also  a  variation  in  the  individual's  resistance  at  different  times 
dependent  upon  food,  sleep,  work  and  general  hygienic  conditions. 

FACTORS  OF  NATURAL  IMMUNITY. — The  natural  immunity  of  any 
individual  to  an  infection  may  be  dependent  upon  several  things  as 
follows: 

The  Protection  A  forded  the  Body  by  the  Surfaces. — The  body  surfaces 
may  be  conveniently  divided  into  those  which  are  external  and  those 
which  are  internal. 

Skin  and  Cutaneous  Orifices.— The,  first  protective  mechanism  that 
we  wish  to  call  attention  to  is  the  skin.  It  is  a  well-known  fact  that 
virulent  bacteria  are  frequently  present  on  the  skin  of  seemingly  normal 
and  healthy  individuals.  Perhaps  the  most  common  of  these  is  the 
Strept.  pyogenes  and  the  M.  pyogenes  vars.  aureus  and  albus.  These 
microorganisms  and  others  live  largely  as  saprophytes,  feeding  upon 


IMMUNITY    AND    SUSCEPTIBILITY  689 

the  dead  and  desquamating  epithelial  cells.  The  skin  is  impermeable 
to  these  microorganisms  when  it  is  unbroken  in  its  normal  state. 
Experiments  have  been  performed  to  determine  whether  the  skin  is 
normally  permeable  to  bacteria.  Bacteria  have  been  rubbed  into  the 
skin  and  have  produced  infection  but  in  these  instances  the  skin  has 
been,  abraded  by  the  mechanical  irritation.  Bacteria  may  infect  the 
sudoriferous  and  sebaceous  glands  and  their  ducts,  in  case  the  metabolic 
activity  of  these  structures  is  disturbed.  The  ducts  and  the  glands  of 
the  skin  are  protected  ordinarily  by  a  flow  of  the  secretions.  In  case 
the  flow  of  the  secretions  is  decreased  and  the  orifices  of  the  ducts 
contracted  as  in  cold  weather,  while  bacteria  find  it  more  difficult  to 
pass  down  than  before,  they  occasionally  do  produce  an  infection. 
When  a  hair  follicle  is  diseased  and  the  shaft  contracted  or  perhaps 
dropped  out,  bacteria  may  pass  down  and  produce  an  infection.  B. 
tetani  of  tetanus  or  lockjaw  frequently  passes  through  the  skin  by 
means  of  deep  penetrating  wounds.  The  same  is  true  of  some  other 
pathogens. 

In  case  bacteria  are  successful  in  permeating  the  skin  either  directly 
or  by  means  of  cutaneous  orifices,  they  are  usually  able  to  set  up  a 
marked  inflammation  of  these  structures  and  produce  necrosis  of  the 
epithelium.  It  is  in  this  way  that  pustules,  boils,  carbuncles,  and 
various  forms  of  cellulitis  are  produced.  The  secretions  of  the  sebace- 
ous glands  are  not  germicidal  but  are  perhaps  slightly  antiseptic  due 
to  the  salts  which  are  contained  therein.  Furthermore,  as  soon  as  the 
serum  from  the  blood  is  extravasated  there  may  be  slight  germicidal 
action  on  the  bacteria  infecting  the  skin.  The  soluble  toxins  of  bacteria 
cannot  be  absorbed  through  the  unbroken  skin. 

The  Subcutaneous  Tissue. — In  case  the  bacteria  are  successful  in 
permeating  the  skin  and  penetrating  the  subcutaneous  connective 
tissue,  again  various  protective  mechanisms  show  themselves.  This 
resistance  is  due  to  a  very  rapid  production  of  new  connective  tissue 
which  serves  to  mechanically  limit  the  infection.  It  is  due,  further- 
more, to  the  germicidal  action  of  the  serum,  the  mechanical  and  germi- 
cidal action  of  the  fibrin  and  the  phagocytic  activity  of  the  leucocytes. 
These  various  factors  will  be  discussed  subsequently  in  connection 
with  the  phenomena  and  the  protective  mechanisms  of  inflammation. 

The  Exposed  Mucous  Membranes  of  the  Body. — The  exposed  mucous 
membranes  of  the  body  usually  are  covered  with  a  variety  of  bacteria, 

44 


690   MICROBIOLOGY  OF  DISEASES   OF  "MAN  AND  DOMESTIC   ANIMALS 

some  of  which  are  pathogenic.  Their  moist  condition  favors  the  growth 
of  microorganisms,  but  the  mucus  which  is  secreted  upon  them  forms 
a  mechanical  barrier  to  the  bacteria  and  serves  to  wash  them  away. 
This  mucus  is  not  germicidal  but  is  perhaps  slightly  antiseptic.  The 
only  mucous  membranes  of  the  body  that  are  really  exposed  are  those 
of  the  eyelids,  lips,  anterior  nares,  genito-urinary  apparatus  and  the  anus. 
It  is  perhaps  more  convenient  to  discuss  these  membranes  in  detail  in 
connection  with  the  cavities  which  are  connected  with  them. 

Nasal  Cavity. — Microorganisms  find  a  barrier  to  the  entrance  of 
the  nasal  cavity  in  the  hairs  which  protect  the  anterior  nares  and 
serve  to  keep  out  the  dust  from  the  inhaled  air.  The  membranes  of 
the  nasal  tract,  besides  being  covered  with  mucus,  which  acts  as  above 
mentioned,  are  also  covered  with  ciliated  epithelial  cells  which  move 
from  within  out  and  serve  to  wash  the  mucus  containing  the  bacteria 
from  the  surface.  Infections  of  the  nasal  mucous  membranes  are, 
however,  not  uncommon.  Bact.  influenza,  Strept.  pyo genes,  M. 
pyogenes  vars.  aureus  et  albus,  Bact.  diphtheria,  M.  intracellularis  var. 
meningitidis,  and  occasionally  Bact.  mallei  produce  infection  through 
this  membrane. 

The  Mouth. — The  mouth  probably  contains  the  largest  variety  of 
bacteria  to  be  found  anywhere  in  the  body.  A  large  number  of  these 
bacteria  are  non-pathogenic,  although  pathogenic  microorganisms  do 
occasionally  occur.  All  the  requisite  conditions  for  bacterial  growth  are 
provided  in  the  mouth,  namely,  temperature,  moisture  and  food.  The 
food  supply  is  largely  derived  from  materials  which  have  been  de- 
posited during  the  process  of  mastication  between  the  teeth  and  in  the 
various  depressions  of  the  mucous  membrane.  The  microorganisms 
also  feed  upon  the  desquamated  squamous  epithelial  cells.  They  are 
being  constantly  washed  off  the  membrane  by  the  saliva  which  contains 
a  certain  portion  of  mucus.  The  saliva  is  not  germicidal,  and  in  all 
probability  only  very  slightly  antiseptic.  The  most  permeable  part 
of  the  mouth  is  in  all  probability  the  tonsils  which  separate  this  cavity 
from  the  pharynx  or  throat.  These  lymphatic  structures  have  many 
deep  crypts,  and  bacteria  once  entering  the  tissues  of  the  tonsils  may 
gain  access  to  the  lymphatic  circulation  through  these  structures. 

In  case  bacteria  are  successful  in  passing  the  obstacles  of  protection 
afforded  in  the  nose  and  in  the  mouth  and  pass  into  the  throat,  there  are 
two  routes  for  their  entrance  into  the  internal  body,  namely,  through 


IMMUNITY   AND    SUSCEPTIBILITY  691 

te  trachea  and  bronchi  into  the  lungs  and  through  the  oesophagus  into 
ic  stomach  and  intestines. 

The  Lungs. — In  case  infectious  microorganisms  pass  down  the 
trachea  and  bronchi  they  meet  first  with  the  obstruction  of  the  mucus 
which  is  secreted  upon  the  surfaces  of  these  tubes.  In  addition,  ciliated 
epithelial  cells  are  present  and  serve  to  cleanse  the  surfaces  from 
microorganisms  as  in  the  nose.  Occasionally  microorganisms  lodge 
along  the  trachea  and  the  bronchi  and  produce  slight  irritations  which  if 
left  undisturbed  may  immediately  produce  serious  infections.  How- 
ever, the  leucocytes  from  the  neighboring  bronchial  and  mediastinal 
lymph  glands  pass  through  the  walls  of  the  trachea  and  bronchi,  ingest 
the  microorganisms,  carry  them  back  to  the  glands  and  in  a  majority 
of  instances  destroy  them.  Occasionally,  however,  leucocytes  contain- 
ing virulent  microorganisms  get  into  the  lymphatic  circulation  and  these 
are  carried  by  the  diffusion  currents  in  the  lymph  vessels  down  to  the 
alveoli  of  the  lungs  and  here  may  cause  inflammations  of  a  more  or  less 
serious  character.  It  is  probable  that  the  Strept.  pneumonia  is  very 
frequently  carried  to  the  alveoli  of  the  lungs  in  this  way.  Without 
doubt,  microorganisms  cannot  be  directly  inhaled  through  the  air 
passages  into  the  alveoli  of  the  lungs  during  an  ordinary  inspiration, 
but  it  has  been  shown  that  in  forced  inspirations,  such  as  those  attend- 
ing upon  coughing}  hiccoughing,  sneezing  and  sighing  that  they  may  be 
so  carried. 

The  Stomach. — In  case  the  microorganisms  pass  down  the  oesophagus 
into  the  stomach,  they  immediately  come  into  contact  in  the  normal 
organ,  with  the  gastric  juice,  which  contains  the  hydrochloric  acid  in 
such  concentration  that  it  is  at  least  antiseptic  if  not  germicidal. 
In  case  the  functional  activity  of  the  stomach  is  disturbed  and  the 
hydrochloric  acid  is  diminished  in  amount,  microorganisms  may  grow 
in  the  stomach  to  a  limited  extent.  Furthermore,  in  case  all  the 
particles  of  food  are  not  thoroughly  broken  up  in  the  stomach,  bacteria 
which  may  be  contained  within  these  particles  may  pass  through  the 
stomach  into  the  intestine. 

The  Intestines. — In  the  intestines  the  microorganisms  come  into 
contact  with  the  alkaline  pancreatic  juice  which  is  slightly  antiseptic 
and  with  the  bile  which  is  antiseptic  and  in  certain  instances  bacteri- 
cidal. They  find  no  particularly  favorable  conditions  for  growth  in  the 
upper  part  of  the  small  intestines  under  normal  conditions.  Here 


692    MICROBIOLOGY  OF  DISEASES   OF  -MAN  AND  DOMESTIC  ANIMALS 

also  mucus  covers  the  surfaces.  However,  if  the  functional  activity 
of  the  small  intestines  is  disturbed,  bacteria  may  enter  the  lymphatic 
structures  (Peyer's  patches,  solitary  follicles)  low  down  in  the  small 
intestines  and  produce  infection.  Such  is  the  case  with  B.  typhosus 
of  typhoid  fever  and  with  the  Msp.  comma  of  Asiatic  cholera. 
Bacteria  which  have  been  prevented  from  development  in  the  small 
intestines  frequently  find  the  opportunity  in  the  large  intestine. 
Here  the  concentration  of  the  various  digestive  juices  is  lowered  and 
the  requisite  condition  for  maximum  bacterial  growth  is  provided. 
Nevertheless,  infections  of  the  large  intestine  with  bacteria  are  not 
common  but  may  occur,  colitis  of  various  forms  resulting.  The 
various  dysentery  amoebae  very  frequently  develop  in  the  large 
intestine. 

The  Genito-urinary  Tract. — The  mucous  membranes  of  the  genito- 
urinary tract,  varying  in  male  and  female,  present  the  same  features 
as  those  of  other  mucous  membranes.  Besides  the  secretion  of  mucus, 
various  other  acid-containing  secretions  are  often  present.  In  addi- 
tion, in  the  urinary  tract  the  mechanical  factor  of  irrigation  removes  the 
microorganisms.  Not  infrequently,  however,  microorganisms  do 
enter  these  mucous  membranes  and  produce  serious  infections,  such 
as  the  Treponema  pallidum  of  syphilis,  the  M.  gonorrhoea  and  the 
B.  chancroids  mollis.  Sometimes  these  membranes  are  infected  with 
ordinary  pyogenic  bacteria. 

The  Conjunctiva. — The  conjunctiva  is  protected  against  infection 
in  several  ways.  First,  the  eyebrows  with  their  hairs  and  the  eye- 
lashes prevent  microorganisms  and  particles  of  dust  and  dirt  carry- 
ing microorganisms  from  entering  the  eye.  Again,  the  lacrymal 
secretion  or  the  tears  flowing  across  the  eye  from  the  outside  in  serve 
to  wash  this  membrane.  Bacteria  are  frequently  washed  off  the 
conjunctiva  and  pass  down  through  the  lacrymal  duct  into  the  nose 
where  they  meet  the  obstructions  which  have  been  previously  dis- 
cussed. In  all  probability  the  tears  are  only  slightly  antiseptic  and 
not  germicidal  at  all.  The  conjunctiva  is  sometimes  infected  with 
microorganisms  and  furthermore  serves  as  a  point  of  entrance  into  the 
body  when  it  itself  is  not  infected.  There  is  no  doubt  that  the  Bact. 
influenza,  the  Strept.  pneumonia,  and  other  microorganisms  may  enter 
the  body  and  get  into  the  lymphatic  and  blood  circulation  in  this 
way. 


IMMUNITY    AND    SUSCEPTIBILITY  693 

It  is  evident,  therefore,  that  the  protection  afforded  an  individual 
by  the  body  surfaces  is  a  decided  factor  in  the  natural  immunity  of 
that  individual. 

The  Protective  Nature  of  Inflammatory  Processes. — It  has  been 
mentioned  in  a  previous  discussion  that  when  bacteria  successfully 
enter  a  tissue  and  develop  in  that  tissue  a  complex  local  change  results 
which  is  designated  as  inflammation.  In  the  majority  of  instances 
inflammation  is  of  a  beneficial  nature.  Fundamentally,  it  is  always 
beneficial.  Few  examples  of  the  pernicious  results  from  inflammation 
can  be  given.  In  this  connection  may  be  mentioned  the  thickening 
of  the  cerebral  blood-vessels  in  syphilis  and  the  increase  of  connective 
tissue  in  cirrhosis  of  the  liver.  In  these  instances  the  inflammatory 
processes  are  brought  about  by  the  reaction  of  the  various  tissues  to 
the  irritation  of  the  infecting  microorganisms.  Unluckily  these  reac- 
tions are  not  on  the  whole  beneficial  to  the  body,  but,  as  before  stated, 
inflammation  is  usually  beneficial  and  may  be  characterized  as  the  re- 
action of  tissues  to  injury.  The  exact  processes  of  inflammation  may 
be  traced  in  case  an  infecting  microorganism  succeeds  in  entering  the 
tissues  of  the  body.  The  organism  having  produced  its  toxic  substance 
first  causes  a  congestion  of  the  blood-vessels  in  the  region  (hyper- 
emia).  Following  this  localized  congestion  there  is  an  extravasation 
of  plasma  from  the  blood-vessels.  This  plasma  immediately  on  leav- 
ing the  vessels  coagulates  or  clots,  producing  throughout  the  infected 
area  fibrin  and  blood  serum.  This  fibrin  serves  in  a  mechanical  way 
to  limit  the  infection,  and  it  has  been  recently  demonstrated  that 
the  fibrin  possesses  germicidal  properties  in  addition.  Furthermore, 
the  serum  in  a  large  number  of  instances  exerts  a  bactericidal  effect 
upon  the  microorganisms.  Following  the  extravasation  of  blood 
plasma  from  the  capillaries,  the  leucocytes  pass  out  and  gather  around 
the  infected  area.  These  leucocytes  are  attracted  to  the  area  due 
to  the  presence  of  various  chemical  substances  (chemotaxis).  They 
will  come  as  close  to  the  microorganisms  as  possible,  depending  upon 
the  effect  of  the  toxins  which  have  been  produced.  In  certain  in- 
stances they  will  ingest  the  bacteria  and  destroy  them.  In  such 
cases,  the  bacteria  having  been  removed,  the  inflammation  rapidly 
subsides  and  the  infection  is,  therefore,  checked.  Such  are  the  char- 
acteristics of  an  acute  inflammation.  Inflammations,  however,  not 
infrequently  may  become  chronic,  depending  especially  upon  the  mi- 


694   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

croorganism  producing  the  infection,  and  in  such  a  case  the  inflamma- 
tion after  passing  through  the  acute  stage,  as  indicated  above,  stimu- 
lates a  proliferation  of  the  connective  tissue  in  the  part  infected. 
In  such  cases,  around  the  outside  of  the  ring  of  leucocytes,  which 
have  been  unable  to  ingest  the  bacteria,  young  embryonic  connective- 
tissue  cells  which  are  known  as  round  cells  are  found.  In  case  the 
inflammation  progresses,  the  leucocytes  are  destroyed  and  the  round 
cells  next  to  the  infected  area  assume  more  of  an  elliptical  shape  and 
are  known  as  epithelioid  cells.  On  the  outside  of  this  layer  of  epi- 
thelioid  cells  will  be  found  newly  produced  round  cells,  and  on  the 
outside  of  the  round  cells  an  area  of  recently  migrated  leucocytes,  those 
passing  out  in  the  beginning  having  been  destroyed  by  the  toxic  action 
of  the  infecting  microorganisms.  Frequently  the  newly  produced 
connective  tissue  passes  on  to  the  adult  type  and  in  this  instance 
completely  walls  off  the  area  of  infection  and  the  infecting  micro- 
organisms. In  such  cases  the  inflammation  and  the  infection  are 
checked.  Among  the  diseases  caused  by  microorganisms  which  have  a 
tendency  to  produce  chronic  inflammation  may  be  mentioned  those  of 
tuberculosis,  leprosy,  syphilis,  actinomycosis  and  glanders.  It  is  not 
an  uncommon  observation  in  man  to  note  in  the  lungs  and  in  other 
parts  of  the  body  healed  areas  of  tubercular  infection;  areas  that  have 
been  completely  walled  off  by  the  development  of  adult  fibrous  tissue. 
It  is  probable  that  about  ninety-five  per  cent  of  all  individuals  living  in 
civilized  communities  are  infected  with  Bact.  tuberculosis  some  time 
during  their  lives.  The  inflammation  produced  by  this  microorganism 
passes  through  the  acute  stage  and  into  the  chronic  before  being  suc- 
cessfully combated  and  thoroughly  walled  off.  Such  an  area  is  known 
as  a  tubercle,  and  in  the  other  diseases  mentioned,  similar  areas  of  like 
structure  are  produced.  It  depends  entirely  upon  the  virulence  of  the 
infecting  microorganisms,  and  the  resistance  of  the  connective  tissue 
of  the  individual  infected  as  to  whether  healing  will  result. 

Natural  Antitoxins. — It  is  an  observed  fact  that  certain  animals 
resist  the  action  of  toxins  produced  by  bacterial  and  other  plant  and 
animal  cells.  The  question  arises  as  to  whether  these  animals  are 
immune  to  the  toxins  on  account  of  the  presence  in  their  bodies  of 
natural  antitoxins  or  other  substances.  If  antitoxin  is  present,  it 
can  be  detected  by  experiments  made  by  drawing  off  the  blood  serum 
of  the  animal  and  combining  it  in  varying  proportions  with  the  toxin  in 


IMMUNITY   AND    SUSCEPTIBILITY  695 

question.  These  experiments  may  be  made  in  vitro.  When  toxins 
and  antitoxins  are  combined  in  proper  proportion  and  incubated 
together  a  non-toxic  molecule  is  produced  which  when  injected  into  a 
susceptible  animal  will  produce  no  effect.  It  is,  of  course,  necessary 
in  this  connection  to  inject  the  animal  with  a  minimum  lethal  dose  of 
the  toxin  in  question  as  a  control.  If  no  natural  antitoxins  are  present 
in  the  serum  of  the  animal  in  question,  the  animal  experimentally 
injected  with  the  combined  toxin  and  serum  will  die  as  a  result  of  the 
non-combination  of  the  toxin.  In  this  way  natural  antitoxins  may 
be  tested.  Natural  antitoxins  for  diphtheria  have  been  detected  in 
the  blood  serum  of  about  fifty  per  cent  of  normal  humans  and  in  about 
thirty  per  cent  of  horses.  However,  their  occurrence  in  other  animals 
for  this  specific  bacterium  and  for  other  species  is  comparatively  rare, 
and  the  explanation  of  the  fact  that  certain  animals  are  immune  to 
toxins  must  be  found  elsewhere.  It  has  been  shown  for  example  that 
the  frog  is  immune  to  tetanus  toxin,  and  that,  when  this  animal  is 
injected  with  this  toxin,  a  large  part  of  the  toxin  remains  unchanged 
in  the  circulation  for  a  variable  period  of  time  and  may  be  later  drawn 
off  in  the  blood  serum,  producing  a  toxic  effect  when  injected 
into  a  susceptible  animal.  There  are  no  natural  antitoxins  present  in 
the  blood  serum  of  the  frog  and  it  has  been  found  that  the  immunity 
of  this  animal  is  due  to  the  fact  that  there  are  no  cells  in  the  body 
possessing  the  necessary  side-chains  (open  valencies)  for  chemical 
combination  with  the  toxin  and  the  subsequent  intoxication  of  the 
cells  does  not  result.  It  seems  that  the  best  explanation  of  the  fact 
that  certain  animals  are  immune  to  toxins  is  found  in  the  fact  that 
there  are  no  chemical  substances  in  the  cells  with  which  toxin  can 
combine.  It  is  probably  not  true  that  natural  antitoxins  explain  all 
the  phenomena  in  this  connection. 

Natural  Antibacterial  Substances. — Natural  antibacterial  substances 
are  present  in  the  blood  serum  and  body  fluids  of  a  large  number  of 
animals.  In  order  to  demonstrate  the  presence  of  the  natural  anti- 
bacterial substances  it  is  necessary  to  inject  the  experimental  animal 
with  a  carefully  washed  culture  of  the  bacteria  in  question.  If  the 
animal  remains  uninfected,  two  possibilities  present  themselves:  First, 
the  presence  of  natural  antitoxins;  and  second,  the  presence  of  anti- 
bacterial substances.  It  is  necessary,  of  course,  to  have  excluded  the 
possibility  of  natural  antitoxins,  it  having  been  demonstrated  that 


696   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND   DOMESTIC  ANIMALS 

the  organism  injected  produces  its  diseased  effects  by  endotoxins  held 
within  the  bacterial  cell  rather  than  by  toxins.  There  is  no  evidence 
indicating  the  presence  of  natural  antiendotoxins  in  any  animal. 
The  antibacterial  action  of  the  blood  may  be  due  to  two  constituents, 
namely,  cellular  substances  (leucocytes)  and  chemical  substances  in 
the  serum.  The  rat  and  the  dog  are  both  immune  to  anthrax  but  the 
immunity  of  the  dog  is  not  due  to  antibacterial  substances  but  to  the 
phagocytic  activity  of  the  leucocytes,  while  in  the  rat  the  immunity  is 
not  due  to  the  leucocytes  but  to  the  antibacterial  substances.  In 
order  to  demonstrate  the  fact  that  the  leucocytes  are  not  responsible 
for  the  immunity  in  the  given  animal  it  is  necessary  to  combine  the 
bacteria  in  question  with  the  leucocytes  and  serum  in  vitro  and  after 
incubation  make  a  careful  examination  with  these  cells  to  see  if  they 
have  taken  up  any  bacteria. 

Antibacterial  action  is  due  to  two  substances  in  the  serum:  First, 
the  thermostable  substance  which  combines  with  the  bacteria  called  an 
amboceptor;  and  second,  a  thermolabile  substance  called  a  complement, 
which  combines  with  the  amboceptor  after  this  substance  has  combined 
with  the  bacterial  cell.  It  is  sufficient'  to  say  at  this  time  that  these 
substances  occur  in  normal  sera  and  that  the  result  of  their  combination 
with  the  bacterial  cell  causes  the  death  of  the  bacteria  and  in  some  cases 
a  lysis  (solution)  of  the  bacteria  in  addition. 

There  may  be  present  in  the  blood  of  animals  antibacterial  sub- 
stances of  three  kinds:  First,  those  just  killing  bacteria  (bactericidal); 
second,  those  killing  the  bacteria  and  dissolving  them  (bacteriolytic) ; 
and  third,  the  leucocytes  which  are  active  in  the  ingestion  of  the  specific 
microorganisms  and  subsequently  digest  and  destroy  them.  In  all 
probability  the  overactivity  of  leucocytes  in  every  case  of  natural 
phagocytic  immunity  is  due  to  the  presence  of  normal  opsonins — sub- 
stances which  sensitize  the  bacteria  and  render  them  susceptible  to 
phagocytosis. 

Normal  Hemolysins. — Normal  hemolysins  (hemoglobin-liberating 
substances)  are  present  in  the  sera  of  certain  animals  for  the  red  blood 
corpuscles  of  other  animals  of  different  species,  and  for  the  same  species, 
but  never  for  the  red  corpuscles  of  the  animal  from  which  the  serum  was 
obtained.  Such  substances  known  respectively  as  heterolysins  and 
isolysins  and  if  the  latter  occurred  the  name  autolysin  would  be  applied. 

Normal  Agglutinins. — Normal  agglutinins  for  various  bacteria,  such 


IMMUNITY    AND    SUSCEPTIBILITY  697 

as  B.  typhosus,  Msp.  comma,  Bact.  dysenteries,  B.  coli,  and  Ps.  pyocyanea 
are  present  in  the  blood  serum  of  some  animals.  It  is  necessary,  of 
course,  to  exclude  normal  agglutinins  when  testing  the  serum  of  the 
infected  case  for  the  purposes  of  diagnosis  as  will  be  mentioned  later. 

Normal  Precipitins. — No  normal  precipitins  for  bacteria  occur  in  the 
sera  of  animals.  Precipitins  for  various  blood  sera,  however,  do  occur. 
-For  example,  human  serum  will  precipitate  the  serum  of  certain  species 
of  monkeys.  These  substances  will  be  discussed  in  detail  under  ac- 
quired immunity. 

ACQUIRED  IMMUNITY. — Acquired  immunity  is  that  resistance  which 
is  acquired  after  having  an  infection  or  from  artificial  inoculation 
with  the  etiological  microorganism  of  an  infection  or  from  inocula- 
tion with  the  products  remaining  in  the  body  after  infection,  whether 
natural  or  artificial.  Acquired  immunity  may  be  divided  into  two 
classes,  namely,  active  and  passive.  Active  immunity  is  that  immunity 
resulting  from  an  infection  or  vaccination.  In  it  the  body  cells  react 
and  give  rise  to  the  formation  of  antibodies.  When  antibodies  pro- 
duced in  active  immunity  are  inoculated  into  other  animals  the  im- 
munity conferred  is  referred  to  as  passive  immunity. 

Active  Immunity. — Active  immunity  may  be  produced  artificially 
in  the  following  ways:  By  the  injection  of  living  bacteria;  by  the  injec- 
tion of  bacteria  of  reduced  virulence;  by  the  injection  of  dead  bacteria; 
by  the  injection  of  the  secretory  and  excretory  products  of  bacteria 
(toxins,  etc.) ;  by  the  injection  of  the  disintegration  products  of  bacteria 
liberated  after  the  death  of  the  cells  (endotoxins) ;  and  by  the  injection 
of  bacteria  or  bacterial  products  which  in  no  way  are  related  to  the  bac- 
terium against  which  immunity  is  conferred. 

As  a  result  of  the  injection  of  living  bacteria  in  small  amounts  or  of 
bacteria  of  reduced  virulence  the  body  cells  react  and  produce  bacteri- 
cidal substances  (lysins,  etc.).  As  a  result  of  the  injection  of  dead  bac- 
teria, the  opsonins  are  increased  in  the  blood.  As  a  result  of  the  injec- 
tion of  the  secretory  and  excretory  products  of  the  bacteria,  namely, 
toxins,  antitoxins  are  produced.  As  a  result  of  the  injection  of  the  dis- 
integration products  of  bacteria,  namely,  endotoxins,  bactericidal  sub- 
stances are  produced.  In  cases  where  bacteria  or  bacterial  products, 
which  are  in  no  way  related  to  the  bacterium  against  which  immunity 
is  conferred  are  injected,  it  is  probable  that  bactericidal  substances  are 
produced.  This  condition  only  occurs  in  rare  instances. 


698   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

Passive  Immunity. — Passive  immunity  may  be  conferred  by  the 
injection  of  antitoxins,  and  by  the  injection  of  bactericidal  substances. 
In  this  type  of  artificially  produced  acquired  immunity  the  body  cells 
do  not  react  to  any  great  extent  and  the  injected  antibodies  remain 
practically  unaltered.  Various  other  antibodies  may  be  injected  into 
other  animals  and  confer  upon  them  passive  immunity. 

The  principal  antibodies  produced  in  active  immunity  will  be 
subsequently  discussed. 

THE  ORIGIN  AND  OCCURRENCE  OF  ANTIBODIES 

The  toxic  and  some  of  the  non-toxic  substances  of  bacteria  and  cells 
from  other  sources  when  introduced  into  the  body  of  a  susceptible 
animal  usually  have  the  power  to  produce  antibodies.  Substances 
having  the  power  of  producing  antibodies  are  known  as  antigens. 
Among  the  antibodies  produced  are  antitoxins,  bactericidal  and  lytic 
substances,  opsonins,  antiferments,  agglutinins  and  precipitins.  The 
antigenic  substances  for  these  antibodies  will  be  discussed  later.  The 
mechanism  of  action  of  the  antigen  is  of  interest.  It  is  supposed  that 
the  antigen  can  combine  only  with  the  cell  which  has  the  proper  com- 
bining groups  or  receptors.  The  antigen  combines  in  the  same  way  that 
food  products  combine  with  the  tissue  cells.  In  case  there  is  no  group 
in  the  tissue  cell  with  which  the  antigen  can  combine  that  tissue  is 
naturally  immune  to  the  antigenous  substances  in  question.  In  the 
same  way  tissue  cells  cannot  utilize  certain  foods  because  they  have  no 
combining  groups.  If  all  the  tissue  cells  in  the  body  are  in  this  condi- 
tion then  the  individual  may  be  said  to  be  naturally  immune.  It 
occasionally  occurs  that  certain  cells  of  the  body  are  not  susceptible 
to  the  action  of  antigens  at  one  time  while  at  another  they  are  sus- 
ceptible. For  example,  the  red  blood  corpuscles  of  the  young  chick  are 
not  affected  by  the  lysin-toxin  in  spider  poison  while  those  of  the  adult 
are  readily  hemolyzed  (hemoglobin  liberated).  It  also  occurs  in  rare 
cases  that  the  antigen  when  injected  into  an  animal  whose  tissue  cells 
show  no  affinity  for  it  or  no  proper  receptors,  will  remain  in  the  circu- 
lation for  days  and  weeks  without  combining  and  producing  any 
effect.  The  antigen,  for  example,  a  toxin,  can  be  isolated  from  the  blood 
in  such  a  case  in  the  same  concentration  and  form  as  when  it  was  in- 
jected. Some  antigens  have  special  affinities  for  certain  tissues,  as 
for  example,  tetanus  toxin  and  nerve  cells.  In  this  case,  however,  the 


IMMUNITY   AND    SUSCEPTIBILITY  699 

larger  part  of  the  antitoxin  is  produced  by  cells  other  than  those  of  the 
nervous  system.  The  production  of  antibodies  for  antigens  probably 
occurs  in  the  following  way;  the  antigenous  substances  combine  with 
the  cells  utilizing  all  the  available  receptors,  leaving  none  open  for  food 
and  thus  perverting  the  general  metabolism  of  the  cell.  In  such  cases 
there  is  a  regeneration  of  these  chemical  receptors  by  the  tissue  cells 
which  more  than  compensates  for  those  with  which  the  antigen  has 
combined  and  as  a  result  the  cell  discharges  them  (chemical  substances) 
free  into  the  body  fluids. 

The  various  antibodies  are  usually  produced  with  more  avidity 
by  certain  tissues  than  by  others.  Antibody  formation  may  be  of  a 
strictly  local  character  depending  upon  the  point  where  the  antigen  is 
injected.  For  example,  when  abrin  is  placed  in  the  eye,  antiabrin  is 
produced,  but  only  in  the  eye  so  injected.  In  the  majority  of  cases  the 
antibodies  are  produced  in  some  special  tissue  or  tissues  at  a  distance 
from  the  point  of  injection. 

Following  the  injection  of  an  antigen  into  the  body  of  an  animal 
there  is  always  a  decrease  in  the  resistance  of  that  body  and  a  decrease 
in  the  antibodies  produced  followed  in  a  short  time  by  a  marked  increase 
in  their  formation.  The  former  condition  is  spoken  of  as  the  "negative 
phase"  and  the  latter  as  the  "positive  phase." 

Antibodies  may  be  transferred  from  mother  to  young  before  birth, 
but  only  after  fetal  circulation  is  established.  It  has  been  positively 
demonstrated  that  antibodies  are  not  transferred  by  the  ovum  or  the 
spermatozoon  directly.  They  are  only  carried  from  the  blood  of  the 
mother  and  diffused  through  the  placenta  into  the  blood  of  the  fetus. 
It  has,  however,  been  shown  that  the  eggs  of  immunized  chickens  con- 
tain antibodies  occasionally.  This  is  "germ-cell  transmission"  and 
not  true  hereditary  transmission.  The  transferred  immunity  or  anti- 
bodies do  not  remain  over  two  or  three  months  in  the  bodies  of  the 
offspring  after  birth. 

ANTITOXINS. — Antitoxins  are  so  called  because  they  combine  with 
and  render  inert  the  soluble  toxins.  Antitoxins  are  produced  for  all 
the  bacteria  producing  soluble  toxins  and  for  the  toxic  substances  of  a 
large  number  of  other  plant  and  animal  cells.  Antitoxins  are  the  free 
chemical  receptors  of  certain  of  the  cells  of  the  body.  That  is,  they  are 
chemical  substances  which  have  been  thrown  off  from  the  cells  of  the 
body  and  in  all  probability  were  normally  used  for  the  purpose  of  taking 


700   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

up  food  substances  although  this  is  not  positively  known.  These 
chemical  substances  are  produced  in  excess  of  those  actually  needed 
by  the  cell  due  to  a  stimulation  of  the  cells  by  the  toxin.  The  antitoxins 
are  labile  substances  which  cannot  be  analyzed.  They  may  be  simi- 
lar to  euglobulins.  They  are  composed  of  molecules  of  large  size. 
Antitoxins  when  present  in  the  body  of  an  animal  are  protective 
and  in  many  cases  curative.  According  to  Ehrlich,  toxins  are  as- 
sumed to  possess  two  chemical  combining  groups,  one  known  as 
the  haptophore  group  which  combines  with  the  cells  and  another 
known  as  the  toxophore  group  which  combines  with  the  cell  after 
the  haptophore  group  has  combined  and  this  produces  an  intoxication 
of  the  cell.  The  haptophore  group  of  the  toxin  molecule  is  thermostable 
(heat  resistant)  and  the  toxophore  group  is  thermolabile  (heat  suscep- 
tible). When  a  toxin  is  injected  into  the  body  of  an  animal,  or  is  pro- 
duced during  the  process  of  an  infection,  the  haptophore  group  combines 
with  the  cells  with  which  it  has  an  especial  affinity  and  with  the 
receptors  (chemical  substances  which  are  unsaturated  and  open  to 
combination  with  other  chemical  substances)  of  these  cells.  The 
chemical  receptors  of  the  cells  with  which  the  toxin-haptophore  group 
combines  are  designated  as  haptophile  receptors.  It  is  probable  also  that 
the  toxophore  group  of  the  toxin  combines  with  other  chemical  receptors 
in  the  cell  after  the  haptophore  and  haptophile  groups  have  combined. 
These  are  designated  as  toxophile  receptors.  The  haptophore  receptors 
of  the  toxin  having  combined  with  the  haptophile  receptors  of  the  cells, 
the  toxophore  group  of  the  toxin  then  combines  and  intoxicates,  stimu- 
lates or  sometimes  kills  the  cells  depending  on  the  affinity  for  the  cells 
and  the  concentration  of  this  group.  In  case  the  cell  is  not  killed,  it 
is  stimulated  and  begins  after  a  time  to  return  to  its  normal  functions. 
All  the  available  receptors  of  the  cells  having  been  occupied  and  com- 
bined with,  the  cell  sets  about  to  generate  new  chemical  receptors  in 
order  that  food  substances  and  other  chemical  substances  may  be  taken 
up.  The  cells  produce  these  haptophile  receptors  in  excess,  that  is, 
there  is  over-compensation,  and  they  are  subsequently  excreted  into  the 
lymph  and  blood.  These  haptophile  receptors  are  in  fact  the  chemical 
substances  which  we  know  as  antitoxins.  It  is  not  only  the  cells  with 
which  both  the  haptophore  and  toxophore  groups  of  the  toxin  combine 
because  of  special  affinity,  which  make  all  the  antitoxin,  but  cells  which 
are  widely  separated  from  those  which  have  an  especial  affinity  for  the 


IMMUNITY   AND    SUSCEPTIBILITY  7OI 

toxin,  also  produce  antitoxin.  For  example,  tetanus  toxin  has  an 
especial  affinity  for  nerve  tissue  but  this  tissue  produces  little  of  the 
antitoxin.  In  this  case  most  of  the  antitoxin  seems  to  be  produced  in 
the  spleen,  lymph  glands  and  bone  marrow.  The  haptophore  groups 
of  the  toxin  have  at  least  combined  with  these  cells  and  stimulated  them 
to  the  overproduction  of  haptophile  receptors. 

It  has  been  mentioned  that  the  antitoxins  are  protective  to  the  body 
infected.  The  haptophile  receptors  (antitoxins)  before  they  are  thrown 
off  combine  with  the  toxin-haptophore  and  often  the  toxophore  group 
does  not  have  the  opportunity  for  combining  and  killing  the  cells.  This 
is  in  case  there  is  no  special  affinity  for  the  cells,  as  in  the  above-men- 
tioned chief  antitoxin-producing  cells  in  tetanus.  In  such  cases  fre- 
quently all  the  available  toxin  is  bound  and  very  little  is  left  to  combine 
with  the  tissue  with  which  it  has  an  especial  affinity,  as  is  the  case  with 
tetanus  toxin  and  nerve  tissue.  The  antitoxins  serve  in  this  instance 
as  protective  substances.  Furthermore,  in  case  the  antitoxin  is  ex- 
creted into  the  blood  and  lymph  it  serves  in  addition  as  a  curative  agent, 
all  the  toxin  which  is  produced  combining  with  all  the  available  anti- 
toxin in  the  circulation  and  none  is  left  to  combine  with  the  cells  of 
the  body.  The  maximum  affinity  is  always  between  toxin  and  anti- 
toxin rather  than  between  toxin  and  cell,  if  there  is  any  antitoxin 
present.  Antitoxins  are  prepared  artificially  and  used  for  both  pro- 
phylactic and  curative  purposes  in  the  treatment  and  prevention  of 
certain  of  the  infectious  diseases  such  as  tetanus  and  diphtheria. 

Antitoxins  are  also  produced  in  the  bodies  of  animals  which  are  to 
all  appearances  immune  to  the  toxins  concerned.  For  example,  the 
alligator  is  immune  to  tetanus  but  when  tetanus  toxin  is  injected  into 
this  animal  tetanus  antitoxin  will  be  produced.  In  this  case  the  hapto- 
phore group  of  the  toxin  has  combined  with  certain  of  the  cells  of  the 
body,  but  with  such  cells  as  give  no  opportunity  for  the  toxophore 
group  to  combine,  or  have  no  affinity  for  this  group.  In  the  case  of 
the  alligator  the  nerve  tissue  seems  to  possess  no  chemical  receptors  for 
the  toxin. 

There  are  certain  animals  which  are  very  susceptible  to  the  action 
of  certain  toxins  and  which  will  not  produce  antitoxin  when  the  toxin  is 
injected.  For  example,  the  guinea-pig  and  the  rabbit  will  not  produce 
tetanus  or  diphtheria  antitoxin  when  injected  with  small  and  gradually 
increasing  doses  of  tetanus  or  diphtheria  toxin.  If  the  toxin  is  modified 


7O2    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

chemically  by  the  addition  of  chemicals  such  as  terchloride  of  iodine  or 
by  heat  these  animals  may  be  immunized  and  will  produce  antitoxin. 
In  this  instance  the  virulence  of  the  toxophore  group  is  reduced  and  it 
is  possible  to  inject  the  animals  with  more  toxin,  thus  combining  with 
more  cells  and  finally  liberating  more  antitoxin. 

It  should  also  be  noted  that  animals  of  the  same  species  vary  in 
their  power  to  produce  antitoxin.  The  production  of  the  product 
varies  with  the  age  and  general  condition  of  the  animal  and  with  the 
duration  and  the  degree  of  toxicity  of  the  toxin  used.  On  account 
of  this  condition  it  is  necessary  to  establish  units  or  standards  for 
determining  the  strength  of  antitoxins. 

As  stated  in  the  discussion  of  natural  immunity  to  toxins,  there 
are  some  animals  which  when  injected  with  toxins  do  not  possess  cells 
which  have  receptors  open  for  chemical  combination  and  as  a  result 
the  toxin  remains  free  in  the  circulation  for  varying  periods  of  time. 
For  example,  as  before  stated,  the  frog  is  immune  to  tetanus  and  an 
injection  of  toxin  will  not  produce  any  antitoxin.  If  tetanus  toxin 
is  injected  into  this  animal  it  will  remain  in  the  circulation  in  the 
same  form  as  injected  and  can  be  withdrawn  after  a  few  weeks  or  a 
month. 

The  Mechanism  of  the  Neutralization  of  Toxin  by  Antitoxin.— 
At  one  time  it  was  supposed  that  the  antitoxin  was  but  a  toxin  in  a 
little  different  form  but  this  has  been  absolutely  disproven.  The 
amount  of  antitoxin  produced  is  much  greater  than  the  amount  of 
toxin  which  is  injected  or  produced  during  an  infection. 

The  union  between  toxin  and  antitoxin  is  of  a  definite  chemical 
nature.  After  these  two  substances  unite  the  resulting  compound  is 
absolutely  harmless  and  differs  from  both  the  toxin  and  the  antitoxin 
in  that  it  is  much  more  stable. 

In  the  beginning  all  experiments  dealing  with  the  union  of  toxin 
and  antitoxin  were  performed  in  the  body  of  an  experimental  animal 
(in  vivo)  but  finally  Ehrlich  showed  that  they  would  act  and  combine 
equally  well  in  the  test-tube  (in  vitro)  and  could  be  studied  much  more 
easily. 

The  various  toxins  are  neutralized  by  their  antitoxins  with  varying 
rapidity.  The  concentration  of  these  chemical  substances,  the  tem- 
perature, the  character  of  the  medium  in  which  they  are  placed,  and  the 
amount  of  electrolytic  salts  present,  are  accountable  for  the  differences 


IMMUNITY   AND    SUSCEPTIBILITY  703 

in  length  of  time  of  combination.  In  the  main  these  substances  act 
like  most  chemicals  and  some  of  them  show  evidences  of  following  the 
laws  of  multiple  proportions.  As  a  matter  of  fact  the  same  laws  which 
govern  the  union  of  toxin  and  antitoxin  govern  other  antibodies  and 
their  antigens. 

As  before  stated,  toxins  have  a  greater1  affinity  for  the  free  hapto- 
phile  receptors  of  cells  (free  antitoxin)  than  for  those  still  associated 
with  the  cells.  Toxin  and  antitoxin  will  always  combine,  if  the 
opportunity  presents  itself,  before  toxin  and  body  cells  will  enter  into 
chemical  union.  Furthermore,  in  certain  instances,  such  as  in  diph- 
theria, when  the  toxin  has  been  partially  bound  by  the  body  cells  and 
antitoxin  is  produced  in  sufficient  quantities  or  is  injected,  the  toxin- 
cell  chemical  union  will  be  broken  up  and  the  toxin  and  antitoxin  will 
combine.  Obviously,  antitoxins  of  this  kind  are  very  valuable  in 
effecting  a  cure  in  certain  infections.  In  the  above-mentioned  cases, 
the  union  between  the  toxin  and  the  cell  is  comparatively  unstable  but 
this  is  not  true  in  every  case,  as  for  example,  in  tetanus  or  lockjaw. 
In  this  case  when  once  the  toxin  is  combined  with  the  cells  of  the  nervous 
system  and  other  body  cells  it  is  very  difficult  to  break  up  the  chemical 
combination  by  the  addition  of  antitoxin.  It  requires  exceedingly 
large  doses  and  these  rarely  act  efficiently.  The  union  between  toxin 
and  body  cells  in  this  instance  is  very  stable.  We  have  here  an  ex- 
planation why  tetanus  antitoxin  is  of  so  little  use  for  therapeutic 
purposes.  It  is,  however,  of  use  as  a  prophylactic  when  free  toxin  is 
being  produced  in  the  body.  Diphtheria  antitoxin  is  efficient  both  as  a 
curative  and  prophylactic  agent  for  the  reasons  which  have  been 
discussed  above. 

Antitoxin  like  toxin  is  fairly  unstable  and  such  agents  as  heat, 
light,  and  chemicals,  affect  it  and  reduce  the  potency.  It  may,  how- 
ever, be  dried  and  kept  for  long  periods  of  time  in  the  dark.  It  is 
necessary  in  the  commercial  preparation  of  antitoxin  and  in  its  ex- 
perimental study  to  have  a  unit  or  standard  of  measurement. 

Units  of  Antitoxin. — In  order  to  arrive  at  a  standard  it  is  necessary 
to  accurately  test  a  given  antitoxin  to  determine  the  number  of  so- 
called  antitoxic  units  it  contains. 

In  the  accurate  study  of  the  neutralization  of  the  toxin  by  the 
antitoxin  it  is  noted  that  adding  fractional  amounts  of  the  antitoxin 


704   MICROBIOLOGY   OF  DISEASES   OF.  MAN   AND   DOMESTIC   ANIMALS 

to  the  L°  dose  of  the  toxin*  and  injecting  the  resulting  mixture  into 
test  animals  (guinea-pigs),  there  is  not  a  corresponding  decrease  in  the 
toxicity  as  would  be  expected.  The  toxin  is  made  up  of  various  parts. 
The  part  just  mentioned  has  a  great  affinity  for  the  antitoxin  but  is  not 
really  toxic.  Such  parts  of  the  toxin  molecule  are  called  protoxoids. 
The  protoxoids  compose  about  one-fourth  of  the  amount  of  toxin 
necessary  to  saturate  one  immunity  unit.  After  the  one-fourth 
antitoxin  is  added  to  the  L°  dose  of  toxin  the  mixtures  of  toxin-anti- 
toxin become  less  toxic  for  the  experimental  animals  down  to  the  point 
where  approximately  three-fourths  of  the  amount  of  toxin  necessary 
to  saturate  one  unit  of  antitoxin  has  been  used  (three-fourths  of  L° 
dose).  This  fraction  is  true  toxin.  The  toxicity  of  the  mixture  does 
not  decline  from  this  point  when  antitoxin  is  added  up  to  one  im- 
munity unit,  and  it  has  been  demonstrated  by  Ehrlich  and  others  that 
this  is  due  ^o  another  part  of  the  toxin  molecule  which  has  a  lesser 
affinity  for  the  antitoxin  than  the  true  toxin  itself  and  the  protoxoid. 
This  part  of  the  molecule  is  called  an  epitoxoid,  true  toxoid,  or  toxon. 
The  toxin  molecule  necessary  to  saturate  one  unit  of  antitoxin  is, 
therefore,  made  up  of  one-fourth  protoxoid,  one-half  true  toxin,  and  one- 
fourth  epitoxoid,  true  toxoid  or  toxon.  The  toxon  is  in  certain  instances 
slightly  toxic  and  is  supposed  by  some  to  be  a  secondary  toxin  and  in 
certain  diseases  such  as  diphtheria,  this  substance  has  a  weak  affinity 
for  antitoxin  and  is  a  possible  cause  of  diphtheritic  paralysis. 

Antitoxins  may  be  prepared  for  all  the  bacteria  producing  soluble 
toxins,  such  as  Bact.  diphtheria,  B.  tetani,  B.  botulinus  and  Ps.  pyo- 
cyanea.  Antitoxic  substances  may  also  be  made  for  some  of  the  products 
of  other  bacteria  such  as  the  Strept.  pyogenes  but  these  differ  from  true 
antitoxins.  Antitoxins  may  also  be  prepared  for  the  toxins  of  certain 
plant  cells,  such  as  abrin,  recin,  crotin,  and  for  the  toxins  of  animals, 
such  as  snake  venom  and  spider  poison.  These  substances  are  in 
the  main  similar  to  those  produced  by  bacteria,  although  in  certain 
characteristics  they  differ  materially. 

LYSINS  AND  BACTERICIDAL  SUBSTANCES. — Under  the  lysins  will  be 
discussed  those  substances  occurring  in  normal  and  immune  sera 
which  have  the  power  of  destroying  and  disintegrating  bacteria,  those 
disintegrating  and  liberating  the  hemoglobin  of  erythrocytes  (red 

*  The  amount  which  just  neutralizes  one  unit  of  standard  antitoxin  (United  States  Public 
Health  Service). 


IMMUNITY   AND    SUSCEPTIBILITY  705 

blood  corpuscles)  and  those  substances  which  have  a  lytic  action  on 
various  body  cells.  The  substances  which  act  on  the  bacteria  are 
called  bacteriolysins,  those  acting  on  erythrocytes  are  called  hemolysins, 
and  those  acting  on  the  other  body  cells  are  called  cytolysins.  The 
mechanism  of  these  lytic  processes  is  quite  complex.  It  should  also 
be  noted  in  this  connection  that  there  are  certain  substances  which 
kill  or  seriously  injure  bacteria  and  body  cells  and  do  not  actually 
disintegrate  them.  Such  chemical  bodies  are  designated  respectively 
as  bactericidal  substances  and  cytotoxins. 

The  first  observations  in  regard  to  bactericidal  and  bacteriolytic 
substances  were  made  by  Nuttall  and  later  by  Biichner.  Biichner 
noted  these  substances  in  normal  sera  and  other  body  fluids  and 
named  them  alexins  (Gr.  to  guard).  He  assumed  that  they  were 
concerned  in  the  immunity  of  the  body.  This  is  not  necessarily  true 
as  certain  blood  sera  are  frequently  highly  bactericidal  when  the  fin- 
dividual  is  relatively  susceptible.  This  is  true  of  human  blood  serum 
and  B.  typhosus.  Furthermore,  in  certain  instances  the  animal  is 
immune  to  the  disease  and  the  serum  is  not  in  any  sense  bactericidal. 
This  is  the  case  with  the  dog  and  Bact.  anthracis. 

PfeifTer  a  number  of  years  ago  observed  that  when  Msp.  comma  of 
Asiatic  cholera  was  introduced  into  the  peritoneal  cavity  of  the  normal 
guinea-pig  the  bacteria  underwent  lysis.  He  also  noted  that  the 
process  was  much  more  rapid  in  the  immune  guinea-pig.  Pfeiffer  had 
the  idea  in  the  beginning  that  lysis  did  not  take  place  anywhere  but  in 
the  body  of  the  animal  but  later  it  was  demonstrated  by  a  number  of 
men,  among  them  Metchnikoff,  that  the  lytic  action  would  also  take 
place  in  the  test-tube  (in  vitro). 

Bordet  and  others  later  showed  that  some  normal  sera  possess 
the  power  of  liberating  the  hemoglobin  in  red  blood  corpuscles.  It 
was  also  shown  that  these  hemolytic  substances  could  be  developed  in 
the  body  of  an  animal  if  that  animal  were  injected  or  immunized  with 
a  suspension  of  erythrocytes.  The  phenomenon  of  hemolysis  is  easily 
observed  and  studied  and  the  amount  of  the  hemolytic  agent  can  be 
accurately  determined  as  the  amount  of  hemoglobin  liberated  varies 
accordingly.  The  mechanism  of  hemolysis  and  bacteriolysis  corre- 
spond exactly  and  accordingly  much  about  the  latter  process  was  first 
worked  out  by  experimentation  with  hemolysins. 

Lytic  substances  can  be  prepared  for  a  large  number  of  bacteria  and 

45 


706    MICROBIOLOGY   OF  DISEASES   OF   MAN   AND   DOMESTIC  ANIMALS 

for  many  body  cells,  as  before  stated.  These  substances  may  be 
markedly  increased  by  the  usual  processes  of  immunization.  Those 
substances  which  have  the  power  to  produce  lysins  are  called  lysinogens 
and  are  distinct  antigens.  The  lysins  are  antibodies.  The  lysins  may  be 
prepared  by  injecting  the  experimental  animal  with  the  live  cells,  the 
dead  cells,  the  disintegration  products  of  cells  and  in  some  cases  with  the 
metabolic  products  of  cells. 

The  Structure  of  Lysins. — Lysins  and  bactericidal  substances  have 
been  shown  to  be  composed  of  two  distinct  parts:  one  a  thermolabile 
part  known  as  the  complement  which  is  destroyed  at  a  temperature  of 
55°  to  60°  for  thirty  minutes;  and  another  part  which  is  thermo- 
stable, known,  on  account  of  its  double  combining  ability,  as  an  ambocep- 
ior.  This  amboceptor  will  withstand  heating  to  60°  for  twenty-four 
hours  but  if  the  temperature  is  raised  to  70°  it  is  readily  destroyed.  If 
kept  at  ordinary  room  temperature  or  in  the  ice  box  amboceptors  will 
remain  active  for  years.  According  to  Ehrlich,  amboceptors  are  the 
free  chemical  receptors  of  the  body  cells.  They  are  produced  in  the 
same  way  as  antitoxins  but  differ  from  these  bodies  in  that  they  have 
two  combining  groups,  one  known  as  the  cytophile  group  with  which 
the  amboceptor  combines  with  the  bacteria  or  other  cells,  and  the  other 
known  as  the  complementophile  group,  with  which  it  combines  with 
the  complement.  The  complement  seems  to  be  a  normal  constituent 
of  the  blood  serum  and  other  body  fluids.  It  is  undoubtedly  produced 
by  the  various  body  cells  (leucocytes  et  al.)  and  during  the  immuniza- 
tion of  animals  with  certain  antigens  it  is  probably  increased  only 
slightly,  if  any,  in  amount.  The  complement  is  supposed  to  be  com- 
posed of  two  groups  also,  one  a  haptophore  with  which  it  combines 
with  the  amboceptor,  and  another  a  zymophore  which  readily  produces 
the  lytic  action  after  the  haptophore  has  combined  with  the  ambocep- 
tor. On  heating  the  complement  the  zymophore  group  is  destroyed 
and  a  complementoid  is  produced.  This  substance  is  similar  to  a  toxoid 
and  will  combine  with  amboceptor  but  no  lysis  will  result.  It  is, 
however,  the  amboceptor,  or  so-called  immune  body,  that  undergoes 
the  decided  increase  during  the  processes  of  immunization.  It  can  be 
accurately  demonstrated  that  the  amboceptor  must  combine  with  the 
cell  in  question  before  the  complement  can  combine.  Cells,  such  as 
bacteria  or  erythrocytes,  may  be  saturated  with  amboceptor  and 
washed  and  when  the  complement  is  added  and  combined,  lysis  takes 


IMMUNITY   AND    SUSCEPTIBILITY  707 

place.  The  complement  will  not  combine  with  the  cells  under  any 
circumstances  unless  amboceptor  is  present  and  has  first  combined 
with  the  cells.  It  is  probable  in  a  given  serum  or  body  fluid  that  there 
are  several  complements  which  may  activate  a  variety  of  amboceptors. 
However,  it  has  been  shown  that  the  same  complement  will  activate 
a  variety  of  amboceptors  of  certain  kinds. 

While  the  majority  of  lytic  sera  are  thermolabile  some  have  been 
noted  which  are  thermostable  to  a  certain  degree.     Hamilton  has  de- 
scribed such  a  serum  resulting  after  immunizing  animals  to  Bact.' 
pseudodiphthericB  and  Horton  has  noted  thermostable  substances  in 
normal  rat  serum  which  are  lytic  for  Bact.  anthracis. 

Various  sera  have  Seen  noted  which  possess  amboceptors  for 
certain  cells  but  are  not  lytic  because  they  do  not  possess  the  necessary 
complement.  For  example,  the  serum  of  the  dog  contains  amboceptors 
for  Bact.  anthracis  but  no  complement.  If  in  this  instance  a  foreign 
complement  such  as  that  in  guinea-pig  or  rabbit  serum  is  added  there 
will  be  lysis  of  the  bacterial  cells. 

Occasionally  the  absence  of  complement  is  of  benefit  to  the  animal 
in  question  and  may  account  for  the  seeming  natural  immunity.  For 
example,  the  venoms  of  the  poisonous  snakes  are  nothing  more  than 
amboceptors  and  when  these  substances  are  injected  into  an  animal 
body  such  as  a  hog,  which  does  not  possess  the  required  complement, 
no  lysis  of  the  body  cells  takes  place.  On  the  other  hand,  should  the 
animal,  such  as  a  rabbit  or  man,  possess  the  necessary  complement,  as 
they  do,  lysis  will  take  place. 

Substances  are  sometimes  present  normally  in  sera  which  have 
the  power  of  combining  with  the  amboceptors  which  may  be  present, 
and  prevent  the  latter  from  combining  with  the  cells  so  that  when  the 
complement  is  added  there  will  be  -no  lysis.  Such  substances  must  be 
designated  as  antiamboceptors.  These  antiamboceptors  (antiantibodies) 
may  be  developed  in  an  animal  by  immunization  with  amboceptors 
of  definite  kinds.  There  are  other  substances  which  may  also  engage 
the  amboceptors  which  cannot  be  called  amboceptors  in  the  true  sense 
but  they  accomplish  the  same  purpose  and  are,  therefore,  classed  with 
these  bodies. 

The  Deviation  of  the  Complement. — The  complement  may  be  deviated 
in  several  ways  and  as  a  result  lysis  of  the  cells  in  question  may  be 
prevented. 


708    MICROBIOLOGY   OF  DISEASES    OF   MAN  AND   DOMESTIC  ANIMALS 

Occasionally  there  is  noted  in  sera  normally  substances  which 
may  combine  with  the  complement  and  prevent  this  body  from  com- 
bining with  the  amboceptor.  Such  substances  are  called  anticomple- 
ments  and  may  be  produced  artificially  by  the  immunization  of  animals 
with  complement.  Occasionally  complement  is  absorbed  by  tissue 
cells  and  prevented  from  combining  with  amboceptor.  In  case  there  is 
an  excess  of  amboceptors  in  a  serum  and  only  a  small  amount  of  com- 
plement, it  may  be  deviated.  In  this  case  the  cells  will  have  taken  up 
all  the  possible  amboceptor  and  there  will  be  an  abundance  of  ambo- 
ceptor free  in  the  serum.  It  has  been  demonstrated  that  complement 
will  combine  with  free  amboceptor  before  it  will  combine  with  the 
amboceptor  which  has  been  bound  to  the  cells.  *  In  this  case  all  the  avail- 
able complement  will  be  taken  up  by  the  amboceptor  which  is  free  and 
consequently  there  will  be  no  lysis.  This  fact  is  of  importance  in  certain 
infections  where  the  development  of  bacteriolytic  substances  are  of 
importance  and  necessary  in  effecting  a  recovery.  The_infectious 
microorganisms  may  not  be  destroyed  for  the  above  reason. 

The  Deflection  of  the  Complement  as  a  Test  for  Antibodies. — A  very  in- 
genious procedure  has  been  devised  for  the  testing  of  sera  for  unknown 
antibodies  similar  to  bactericidal  substances  and  lysins.  The  method 
of  demonstrating  the  fixation  of  the  complement  was  first  worked  out 
by  Bordet  and  Gengou.  The  reaction  is  made  use  of  in  the  test  for 
syphilis  which  is  briefly  stated  as  follows:  when  the  syphilitic  antigen 
is  combined  with  the  supposed  amboceptor  in  the  blood  serum  of  the 
suspected  case  of  syphilis  and  a  foreign  complement,  which  has  been 
accurately  standardized,  is  added,  this  complement  is  bound  and  is, 
therefore,  prevented  from  combining  with  red  blood  corpuscles,  and 
a  hemolytic  amboceptor  which  may  be  added  later.  Hemolysis  is, 
therefore,  prevented.  The  general  technic  of  the  test  is  as  follows: 
the  syphilitic  antigen  is  prepared  by  making  an  aqueous  or  alcoholic 
extract  of  the  liver  of  syphilitic  fetus  or  in  several  other  ways.  This 
antigen  is  supposed  to  contain  protein  and  other  chemical  substances 
produced  by  the  Treponema  pallidum,  the  etiological  microorganism 
of  syphilis  or  similar  substances  to  those  produced  by  this  micro- 
organism. The  blood  serum  of  the  suspected  case  of  syphilis  is  heated 
to  56°  for  thirty  minutes  in  order  that  the  normal  or  immune  serum 
complement  may  be  destroyed.  The  new  complement  is  supplied 
from  normal  guinea-pig  serum.  Before  beginning  the  test  it  is  neces- 


IMMUNITY  AND    SUSCEPTIBILITY  7OQ 

sary  to  have  a  rabbit  immunized  with  some  hemolytic  antigen,  such  as 
sheep  or  human  erythrocytes.  There  is  developed  in  the  serum  of  the 
rabbit  the  hemolysin  for  sheep  or  human  corpuscles  which  when  com- 
bined with  these  corpuscles  will  cause  a  liberation  of  hemoglobin. 
In  the  rabbit  serum  there  are  both  hemolytic  amboceptors  and  comple- 
ment. It  is  necessary  to  heat  this  hemolytic  rabbit  serum  to  56°  for 
thirty  minutes  in  order  to  destroy  its  complement  and  also  it  is  necessary 
to  find  out  accurately  the  amount  of  guinea-pig  serum  which  will  com- 
plement the  resulting  hemolytic  amboceptor.  This  definite  amount  of 
complement  having  been  determined,  it  is  mixed  with  syphilitic  antigen 
plus  the  syphilitic  amboceptor,  mentioned  above,  and  allowed  to  incu- 
bate for  one  hour  and  thirty  minutes  at  37°.  If  the  serum  is  from  a 
case  of  syphilis  the  antibodies  (amboceptors)  will  be  present  and  com- 
bine with  the  antigen,  and  also  the  guinea-pig  serum  complement.  The 
next  step  in  the  technic  is  to  add  to  the  above-mentioned  mixture  the 
hemolytic  amboceptor  and  its  antigen,  sheep  corpuscles.  If  the  com- 
plement has  been  bound  there  will  be  none  left  to  combine  with  the 
hemolytic  amboceptor  and  no  hemolysis  of  the  sheep  or  human  cor- 
puscles will  result.  If  the  patient's  serum  does  not  contain  syphilitic 
amboceptors  or  antibodies,  the  complement  will  not  be  bound  and 
hemolysis  will  result.  This  test  has  been  designated  as  the  Wasscrmann 
test  on  account  of  the  man  first  working  it  out  in  the  case  of  syphilis, 
and  has  shown  itself  to  be  very  efficient  in  the  diagnosis  of  this  disease 
in  suspected  cases.  Many  modifications  of  this  test  have  been  devised, 
some  of  which  are  very  accurate. 

The  fixation  of  the  complement  may  be  made  use  of  in  the  detection 
of  any  bacterial  antibody,  the  procedure  being  approximately  the  same 
as  above  indicated  and  the  hemolytic  system  used  as  an  indicator  as  in 
the  case  of  syphilis.  The  antigen,  however,  is  different.  When  working 
with  specific  bacteria  a  suspension  of  bacterial  cells  in  0.85  per  cent 
sodium  chloride  solution  constitutes  the  antigen. 

Cytotoxins  and  Cytolysins. — The  names  cytotoxin  and  cytolysin  are 
used  synonymously  and  are  applied  to  those  substances  in  sera  and 
other  body  fluids  which  have  the  power  of  destroying  cells  other  than 
erythrocytes.  In  a  broad  sense  any  substance  destroying  a  cell  would 
be  cytotoxic  but  the  terms  are  usually  applied  in  the  more  limited 
manner,  as  above  indicated. 

Cytotoxins  are  produced  in  the  same  manner  as  other  antibodies. 


710  MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

The  immunization  of  an  animal,  for  example,  with  renal  (kidney)  cells, 
produces  in  the  blood  serum  of  that  animal  a  cytotoxin  for  the  paren- 
chymatous  cells  of  the  kidney.  Cytotoxins  can  be  produced  for  prac- 
tically all  the  parenchymatous  cells  of  the  body.  These  immune  bodies 
are  not  very  specific  and  even  careful  experimentation  leads  to  confusing 
results.  For  example,  when  an  animal  is  immunized  to  kidney  cells 
there  is  produced  in  the  body  of  the  immune  animal  cytotoxins  for 
kidney  cells  and  also  cytotoxins  in  smaller  amounts  for  other  paren- 
chymatous cells  such  as  those  of  the  liver.  In  the  beginning  it  was  sup- 
posed that  the  cytotoxins  would  be  of  value  in  the  study  of  the  physio- 
logical functions  of  organs  and  tissues.  For  example,  a  cytotoxin 
having  been  produced  for  the  thyroid  gland  or  adrenal  gland  it  would  be 
possible  to  inject  this  into  another  animal,  destroy  the  gland,  and  then 
note  the  effect  on  the  body.  It  was  thought  it  might  be  possible  to 
produce  anticytotoxins  which  would  be  able  to  counteract  the  action  of 
those  cytotoxic  substances  which  are  produced  in  the  body  during  the 
course  of  infections.  However,  the  lack  of  specificity  of  the  cytotoxin 
renders  these  procedures  only  theoretically  possible.  The  fact  that 
cytotoxins  are  produced  for  cells  other  than  those  used  in  the  process  of 
immunization  indicates  that  there  are  similar  chemical  substances  in  the 
various  cells. 

There  are  autocytotoxins  produced  in  the  body.  These  probably 
result  from  the  absorption  of  the  products  of  disintegrated  tissue  cells. 
If  no  anticytotoxins  for  these  autocytotoxins  are  produced,  or  they  are 
not  destroyed  in  some  way,  a  very  "vicious  cycle"  would  result  in 
that  more  of  the  specific  cells  of  the  organ  or  tissue  used  would  be  de- 
stroyed. Cytotoxins  have  been  prepared  for  leucocytes  and  these 
substances  are  sometimes  developed  during  the  progress  of  an  in- 
fection. The  leucocytotoxins  have  perhaps  been  studied  more  than 
any  one  of  others. 

When  ova  are  used  for  the  purpose  of  producing  cytotoxins,  besides 
producing  these  substances  in  the  serum  of  the  immune  animal, 
cytotoxins  for  spermatozoa  of  the  same  species  are  also  produced, 
showing  that  these  cells  have  some  chemical  substances  in  common. 

Metchnikoff,  following  his  idea  that  old  age  is  due  to  a  destruction 
of  tissue  by  the  mononuclear  leucocytes,  hoped  that  it  would  be  possible 
to  produce  a  cytotoxin  for  these  cells.  It  is  claimed  by  some  that  there 
are  specific  substances  produced  by  the  exhaustion  of  certain  cells, 


IMMUNITY   AND    SUSCEPTIBILITY  71 1 

that  is,  a  toxin  of  fatigue.  Weichardt  has  produced  an  antibody  for 
this  toxic  substance  which  must  be  in  reality  an  anticytotoxin. 

It  has  been  suggested  that  the  cardiac  hypertrophy  in  nephritis  is 
due  to  the  effect  of  a  nephrocytotoxin  on  the  peripheral  blood-vessels 
causing  increased  diastolic  pressure  on  the  heart. 

Another  interesting  substance  has  been  produced  and  this  is  called 
syncytiolysin.  It  is  prepared  by  immunizing  animals  with  placental 
cells.  It  is  claimed  that  this  cytotoxin  produces  on  injection  symptoms 
similar  to  those  noted  in  eclampsia  and  it  has  been  suggested  that  the 
production  of  such  a  body  in  the  pregnant  woman  from  the  placental 
cells  may  be  the  cause  of  this  serious  condition.  Liepmann  claims  to 
have  demonstrated  placental  constituents  in  the  blood  of  pregnant 
women  by  means  of  the  precipitin  test.  These  bodies  must  be  the 
antigen  of  cytotoxins.  He  states  that  when  the  blood  of  the  pregnant 
woman  is  mixed  with  the  specific  syncytiolysin  produced  by  im- 
munizing an  animal  with  human  placenta  a  precipitate  occurs.  He 
suggests  the  possibility  of  a  serum  test  for  pregnancy.  Abderhalden 
has  reported  some  interesting  results  with  the  serum  test  for  preg- 
nancy and  cancer.  His  findings  cannot,  however,  be  regarded  as 
conclusive. 

Cytotoxins  are  similar  to  bacteriolysins  and  hemolysins.  They 
consist  of  amboceptors  which  are  activated  by  the  complement  which  is 
normally  present  in  the  serum  or  other  body  fluids. 

THE  OPSONINS  AND  PHAGOCYTOSIS. — It  was  shown  a  number  of 
years  ago  that  certain  types  of  leucocytes  and  other  body  cells  were 
capable  of  ingesting  bacteria  and  other  plant  and  animal  cells.  The 
mechanism  of  this  process  was  not  known  until  Wright  and  Douglas 
demonstrated  certain  substances  in  the  blood  serum  and  other  body 
fluids  which  have  the  power  of  rendering  the  bacteria  susceptible  to 
phagocytosis.  These  substances  are  known  as  opsonins  (Greek: — I 
prepare  food  for).  The  phenomena  of  the  phagocytosis  depend  almost 
wholly  on  these  special  opsonins.  Leucocytes  which  have  been  washed 
free  from  all  serum  will  not  take  up  bacteria  except  a  few  in  rare  in- 
stances. Bacteria  which  have  been  placed  in  contact  with  blood  serum 
or  other  body  fluids  may  be  thoroughly  washed,  and  then  when  they 
are  placed  in  contact  with  the  leucocytes,  they  will  be  taken  up.  The 
opsonin  reacts  chemically  with  certain  substances  within  the  bacteria, 
and  so  to  speak,  sensitizes  them.  Opsonins  are  present  in  many  normal 


712    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

sera  for  the  various  bacteria.  They  may  be  produced  in  animals 
not  containing  them  by  the  process  of  immunization  with  various 
antigenous  microorganisms.  Opsonins  are  destroyed  at  about  60° 
for  thirty  minutes,  but  there  is  some  variation  among  them.  When 
kept  at  o°  opsonins  will  remain  active  for  several  days,  but  at  a 
temperature  of  the  body,  37°,  after  the  serum  has  been  withdrawn, 
they  rapidly  deteriorate.  Many  opsonins  have  the  structure  of 
agglutinins  and  precipitins,  although  they  bear  some  points  of  re- 
semblance to  antitoxins  and  complements.  They  possess  two  so- 
called  chemical  groups,  a  "combining  group"  by  which  they  enter  into 
chemical  union  with  the  bacteria  and  a  "functional  group"  which 
really  sensitizes  the  microorganism  and  makes  it  phagocy table. 

It  has  been  shown  that  the  opsonins  may  be  increased  in  the 
serum  of  the  animal  or  injected  individual  by  the  injection  of  heated 
(60°)  cultures  of  the  specific  etiological  microorganisms.  Such  sub- 
stances are  called  opsonogens  or  vaccines  (bacterins).  Vaccines  are 
used  to  a  certain  extent  in  the  treatment  of  the  various  pus  infections 
due  to  the  staphylococci  and  also  in  tuberculosis  and  pneumonia.  It 
is  supposed  that  the  opsonins  are  produced  in  the  subcutaneous  tissues 
and  in  the  muscles. 

The  Opsonic  Index. — The  concentration  of  the  opsonins  may  be 
recorded  in  an  individual  in  the  following  ways.  Suppose  the  leu- 
cocytes of  the  infected  individual  take  up  a  certain  number  of  bacteria, 
say  an  average  of  5,  after  counting  50  to  100  polymorphonuclear  leu- 
cocytes. In  this  case  the  phagocytic  index  is  said  to  be  5.  Again, 
suppose  the  leucocytes  of  the  normal  individual  take  up  15  of  the  bac- 
teria in  question,  the  average  after  counting  50  to  100  leucocytes 
being  always  taken.  The  phagocytic  index  in  this  case  would  be  15. 
In  order  to  determine  the  opsonic  index  of  an  infected  individual  the 
phagocytic  index  of  the  normal  individual  is  taken  as  a  denominator 
of  a  fraction  and  the  phagocytic  index  of  the  infected  individual  as  the 
numerator  of  the  fraction.  In  the  above  illustration  this  would  be 
^5>  /^  or  reduced  to  decimals  0.33+.  The  opsonic  index,  it  can  be 
seen,  is  somewhat  of  an  indication  of  the  resistance  of  the  particular 
individual  to  the  infecting  microorganism  in  question.  By  the  use 
of  vaccines  the  opsonic  index  may  be  raised  to  at  least  i.o  or  even 
more,  showing  that  the  leucocytes  are  actively  phagocytic  and  the 
opsonins  increased  in  concentration  in  the  blood  serum.  In  such  a 


IMMUNITY   AND    SUSCEPTIBILITY  713 

case  recovery  would  be  indicated.  When  vaccines  are  injected  in  the 
treatment  of  infections  the  opsonic  index  has  been  shown  to  vary  from 
time  to  time.  Within  a  few  hours  after  the  injection  the  opsonic 
index  falls  below  what  it  was  at  the  time  of  the  injection.  This  lower- 
ing of  the  index  is  known  as  the  "negative  phase."  Following  the 
fall  in  the  index  there  is  a  continuous  rise  to  a  point  equal  to  what  it 
was  in  the  beginning  and  above  this  point.  This  rise  in  the  opsonic 
index  is  known  as  the  "positive  phase."  The  individual  receiving  the 
vaccine  usually  shows  an  increase  in  the  symptoms  during  the  "nega- 
tive phase."  Obviously,  it  is  necessary  not  to  give  a  subsequent  in- 
jection of  vaccine  until  the  patient  is  at  the  height  of  the  "positive 
phase."  This  can  be  best  determined  by  determining  the  opsonic 
index. 

Occasionally  counts  are  made  of  the  number  of  leucocytes  which  are 
actually  taking  up  bacteria,  disregarding  the  number  of  bacteria  within 
the  cells.  The  determination  is  always  made  on  the  basis  of  100  and 
the  per  cent  of  leucocytes  which  are  phagocytic  is  taken  as  the  so-called 
percentage  index.  The  percentage  index  also  gives  an  idea  of  the  re- 
sistance of  the  individual.  It  has  been  shown  that  in  the  practical 
work  of  treating  infections  with  vaccines  it  is  not  absolutely  necessary 
to  determine  the  opsonic  index  or  percentage  index.  The  positive 
and  negative  phase  may  be  determined  fairly  well  by  general  clinical 
observations  on  the  infected  individual.  Virulent  bacteria  are  not 
readily  phagocytized.  For  example,  virulent  streptococci  and  pneu- 
mococci  are  not  phagocytized  as  easily  as  non- virulent  forms.  It. 
seems  in  this  instance  that  there  is  some  toxic  or  poisonous  substance 
produced  by  the  bacteria  that  is  antagonistic  to  the  opsonins  or  perhaps 
an  antiopsonin  is  formed. 

The  presence  of  opsonins  in  the  body  fluids  of  an  animal  is  not 
absolute  proof  that  such  animal  is  highly  resistant  to  infections.  The 
resistance  really  depends  on  the  activity  of  the  phagocytes  and  in 
certain  cases  where  the  opsonins  are  high  in  concentration  the  phago- 
cytes are  not  active.  In  other  cases  the  reverse  is  true  and  in  these 
cases  opsonins  and  phagocytosis  are  of  the  utmost  importance  in  the 
immunity  of  individuals.  For  example,  in  anthrax  the  immunity  of 
the  dog  is  due  to  opsonins  and  phagocytosis,  while  in  the  rat,  although 
opsonins  are  present,  there  is  no  phagocytosis  and  immunity  is  due  to 
antibacterial  substances  in  the  blood  serum.  In  certain  infections, 


714   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

such  as  typhoid  fever,  influenza,  and  uncomplicated  miliary  tubercu- 
losis, there  is  a  deficiency  in  leucocytes  (leucopenia)  and  consequently 
even  if  the  opsonins  were  concentrated  and  the  bacteria  sensitized  there 
would  be  very  little  increase  in  the  immunity  from  these  causes. 

Hemoopsonins. — It  has  been  demonstrated  that  very  frequently 
opsonins  for  red  corpuscles  are  present  in  the  sera  and  body  fluids  of 
animals.  Such  bodies  sensitize  the  red  blood  corpuscles  and  render 
them  susceptible  to  phagocytosis  by  the  polymorphonuclear  leucocytes 
and  the  epithelial  and  other  body  cells.  They  are  designated  as  hemo- 
opsonins.  Occasionally  iso-  and  autohemoopsonins  are  present  in 
normal  sera.  For  example,  in  human  blood  serum,  it  is  probable  that 
the  process  of  red  blood  corpuscle  destruction  which  takes  place  in  the 
spleen  may  be  referred  to  the  action  of  these  types  of  opsonins  and 
various  phagocytic  cells. 

AGGLUTININS. — Agglutinins  are  substances,  present  in  the  blood 
sera  and  body  fluids  of  normal  and  immune  animals,  which 
have  the  power  of  producing  a  clumping  and  sedimentation  of  the 
microorganisms  causing  the  specific  infection  or  used  in  artificial  im- 
munization. The  relationship  of  the  agglutinins  to  the  phenomena  of 
immunity  and  the  other  antibodies  which  are  produced  during  the 
process  of  infection  and  experimental  inoculation  is  not  known.  One 
of  the  first  agglutinins  to  be  observed  was  that  occurring  in  the  blood 
serum  in  cases  of  typhoid  fever  and  the  agglutination  reaction  is  now 
made  use  of  in  the  diagnosis  of  this  disease  (Widal  test}.  Agglutinins 
.are  specific  substances  and  at  high  dilutions  only  cause  a  clumping  of 
the  microorganisms  which  give  rise  to  their  formation  (antigens). 

Normal  Agglutinins. — Agglutinating  substances,  as  above  stated,  are 
frequently  found  in  normal  sera.  In  this  case  no  direct  connection 
between  their  formation  and  specific  microorganisms  can  be  established. 
Normal  human  serum  frequently  contains  agglutinins  for  B.  typhosus, 
B.  coli,  Bact.  dysenteries,  and  occasionally  M.  pyogenes  var.  aureus 
and  Msp.  comma  in  certain  rare  cases.  Agglutinins  for  B.  typhosus 
which  are  present  normally  in  the  serum  may  give  rise  to  confusion 
when  this  test  is'used  for  the  diagnosis  of  typhoid  fever.  It  is,  there- 
fore, necessary  to  dilute  the  serum  of  a  suspected  case  of  typhoid  fever 
at  least  one  to  forty  or  one  to  fifty  times  in  order  to  exclude  the  normal 
agglutinins  and  the  so-called  coagglutinins. 

The  Production  of  Agglutinins. — Agglutinins  may  be  produced  arti- 


IMMUNITY    AND    SUSCEPTIBILITY  715 

ficially  by  the  injection  of  bacteria,  dead  or  alive,  into  the  veins,  sub- 
cutaneous tissues  or  peritoneal  cavity.  In  rare  cases  they  may  be  pro- 
duced by  feeding  the  bacteria,  injecting  them  into  the  air  passages  of 
the  lungs  or  by  rubbing  them  into  the  skin.  It  is  probable  Jhat  the 
highest  concentration  of  agglutinins  results  from  the  injection  of  dead 
bacteria.  It  is,  however,  necessary  that  these  bacteria  be  not  subjected 
to  a  temperature  above  62°.  Many  pathogenic  and  non-pathogenic 
bacteria  form  agglutinins  when  injected  into  the  body.  The  concen- 
tration of  the  agglutinins  produced  varies  greatly.  Very  high  agglu- 
tinating sera  are  noted,  such  as,  for  example,  one  in  one  million  when 
B.  typhosus  is  used  and  one  in  two  million  when  Msp.  comma  of  Asiatic 
cholera  is  used.  Often  two  strains  of  the  same  organism  will  vary 
greatly  in  their  power  to  produce  agglutinins.  Again,  the  concentra- 
tion of  the  agglutinins  in  an  infected  animal  varies  from  day  to  day, 
and  in  order  to  make  an  accurate  observation  it  is  necessary  to  make 
repeated  examinations  on  subsequent  days.  Tor  example,  in  typhoid 
fever  the  agglutinins  one  day  may  be  thirty  times  as  strong  as  on  a 
subsequent  day. 

The  Distribution  of  Agglutinins  in  the  Blood. — As  before  stated,  these 
antibodies  are  found  in  practically  all  the  body  fluids.  They  reach 
their  highest  concentration  in  all  probability  in  the  blood  serum.  In 
certain  cases  they  are  in  high  concentration  in  the  milk.  Agglutinins 
are  also  present  at  times  in  the  sputum,  tears,  and  the  humors  of  the  eye. 

Inherited  Agglutinins. — Agglutinating  substances  may  be  transferred 
from  the  mother  to  the  offspring  in  utero.  It  has  been  frequently 
demonstrated,  for  example,  that  the  offspring  of  mothers  who  have 
recently  recovered  from  typhoid  fever  or  who  are  infected  at  the  time 
of  the  birth,  have  agglutinins  in  the  body  fluids.  The  same  is  true  of 
the  offspring  of  glandered  horses.  Notwithstanding  the  fact  that  the 
milk  is  frequently  rich  in  agglutinins,  these  substances  are  not  trans- 
ferred to  the  offspring  to  any  great  extent  by  this  means. 

The  Substances  Concerned  in  Agglutination. — There  are  two  distinct 
substances  concerned  in  this  reaction,  one  substance  which  is  present 
in  the  serum  or  body  fluids  of  the  infected  or  immune  individual,  and 
other  substances  which  are  present  in  the  microorganisms  which  are 
agglutinated.  The  substance  in  the  serum,  as  before  stated,  is  known 
as  the  agglutinin;  the  substance  (antigen)  in  the  bacteria  or  other  micro- 
organisms is  known  as  the  agglutinogen.  When  agglutinins  and  agglu- 


716   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

tinogens  are  combined  together  a  new  substance  is  formed  which  is 
designated  as  an  agglutinate.  As  to  the  location  within  the  bacterial 
cell  of  this  agglutinogen  (agglutinum)  there  is  some  dispute.  Various 
authorities  have  stated  that  it  is  present  in  the  cell  wall  or  on  the  cell 
wall.  Others  have  held  the  view  that  it  is  located  within  the  cell  proto- 
plasm and  in  certain  instances  in  the  flagella.  Without  doubt,  in 
certain  cases  this  substance  is  excreted  from  the  cell  into  the  surround- 
ing medium,  as  is  shown  by  the  fact  that  when  nitrates  of  bacterial  cul- 
tures are  injected  they  frequently  give  rise  to  the  formation  of  agglu- 
tinins.  This  agglutinogenic  substance  is  specific  and  varies  with  the 
species.  There  are,  however,  very  closely  related  substances  of  this 
character  among  some  groups  of  bacteria.  When  these  agglutinogenic 
substances  are  injected  into  the  animal  they  frequently  give  rise  to 
agglutinins  which  when  combined  with  other  members  of  this  group 
will  produce  agglutination  in  low  dilutions.  Such  a  reaction  and  prop- 
erty is  known  as  "group  agglutination"  and  the  agglutinins  produced 
in  such  a  case  are  known  as  co agglutinins.  For  example,  the  serum  of 
the  patient  suffering  from  typhoid  fever  or  of  a  person  or  an  animal 
immunized  with  B.  typhosus  will  produce  an  agglutination  first  of  B. 
typhosus,  but  in  addition,  an  agglutination  of  E.  coli,  B.  paracoli,  B. 
paratyphosus,  and  B.  enteriditis.  The  agglutination  of  these  last- 
named  organisms,  of  course,  will  not  be  active  except  in  low  dilutions, 
and  in  order  to  exclude  them  satisfactorily  it  is  necessary  to  dilute  the 
serum  to  a  higher  point.  This  phenomena  of  coagglutination  is  due 
to  the  fact  that  there  are  some  chemical  substances  (agglutinogenic) 
within  these  bacteria  which  are  common  to  all  and  which  give  rise  to 
the  formation  of  agglutinins,  which  are  chemically  similar  to  each  other 
in  certain  respects. 

Structure  of  Agglutinins  and  Agglutinogens. — According  to  Ehrlich's 
conception  the  agglutinins  are  composed  of  two  chemical  groups,  a 
haptophile  or  combining  group  with  which  it  combines  with  the  hapto- 
phore  group  of  the  agglutinogen  and  a  zymophorous  or  agglutinophor- 
ous  group  which  actually  produces  the  agglutination.  The  agglutino- 
gen is  also  composed  of  a  combining  group  known  as  the  haptophore 
group  with  which  it  combines  with  haptophile  of  the  agglutinin.  It  is 
probable  that  this  same  haptophore  group  will  combine  also  with  vari- 
ous tissue  cells  and  give  rise  to  formations  of  agglutinins  which  are  really 


IMMUNITY   AND    SUSCEPTIBILITY  717 

free  haptophile  receptors  of  the  tissue  cells  which  have  been  acted  upon 
by  the  agglutinogenic  substance  contained  in  the  bacteria. 

Agglutinoids. — It  is  possible  by  means  of  heat  and  chemicals  to 
destroy  the  zymophorous  group  of  the  agglutinin  leaving  only  the  hapto- 
phile group.  Such  a  substance  is  known  as  an  agglutinoid,  being  similar 
to  a  toxoid.  A  temperature  of  not  to  exceed  60°  to  70°  is  necessary 
to  produce  this  substance.  Agglutinoids  will  combine  with  the  agglu- 
tinogen  of  the  bacteria  but  they  will  not  produce  a  clumping  or  an  agglu- 
tinate. Occasionally  in  some  fresh  sera  substances  are  found  which 
have  a  greater  affinity  for  the  agglutinogen  of  the  bacteria  than  the 
agglutinins  have.  Such  substances  are  designated  as  proagglutinoids 
and  are  in  this  respect  similar  to  protoxoids. 

The  Stages  of  Agglutination. — There  are  two  distinct  stages  of  the 
agglutination  reaction.  Neither  of  these  stages  can  take  place  unless 
some  salts  or  electrolytes  are  present.  Sodium  chloride  is  the  common 
salt  present.  The  first  phase  of  the  agglutination  reaction  is  a  union 
between  the  agglutinin  and  the  agglutinogen  of  the  bacteria.  The 
second  phase  is  the  actual  clumping  of  the  bacteria.  It  is  supposed  that 
in  this  last  phase  the  zymogenic  group  of  the  agglutinin  is  acting. 
In  the  first  phase  the  haptophile  group  of  the  agglutinin  is  combined 
with  the  haptophore  group  of  the  agglutinogen. 

There  are  some  bacteria  that  cannot  be  agglutinated,  as  for  example, 
Bad.  pneumonia  of  Friedlander,  and  in  rare  instances  B.  typhosus 
cannot  be  agglutinated.  It  is  possible,  for  example,  to  grow  B.  typhosus 
at  a  temperature  of  42°  and  cause  it  to  lose  its  power  of  producing 
agglutinins.  Bacteria  may  also  be  modified  chemically  so  that  they 
will  lose  the  power  to  produce  agglutinins. 

Agglutinins  bear  no  relationship  to  bactericidal  substances,  anti- 
toxins, opsonins.or  any  of  the  other  antibodies.  They  are  both  of  use 
in  the  determination  of  species  of  bacteria  when  a  known  agglutinating 
serum  is  used,  and  they  are  also  of  use  in  determining  the  cause  of 
infections  where  a  known  culture  or  agglutinogenic  substance  is  used. 
The  agglutination  reaction  is  used  in  the  diagnosis  of  typhoid  fever, 
paratyphoid  fever,  glanders  and  dysentery. 

Hemoagglutinins. — Agglutinating  substances  are  sometimes  pro- 
duced for  red  blood  corpuscles  when  these  cells  are  used  in  the  immu- 
nization of  an  animal.  Such  agglutinins  when  combined  with  the 
corpuscles  produce  a  clumping  which  is  known  as  hemoagglutina- 


718   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

tion.  The  mechanism  of  the  reaction  is  the  same  as  that  of  bacterial 
agglutinins.  It  is  possible  that  hemoagglutination  is  one  impor- 
tant factor  in  the  production  of  agglutination  thrombi  in  certain 
infectious  diseases  such  as  typhoid  fever. 

PRECIPITINS. — Another  group  of  substances,  which  are  antibodies, 
is  produced  through  the  processes  of  immunization  which  have  not 
been  definitely  connected  with  the  phenomena  of  immunity.  These 
substances  are  known  as  the  precipitins.  Precipitins  may  be  produced 
for  the  protein  substances  of  most  bacterial  cells  and  a  large  variety 
of  other  plant  and  animal  cells,  such  as  blood  serum,  milk  and  grains. 
They  were  first  demonstrated  in  1897  by  Kraus,  who  noted  that  the 
bouillon  filtrates  of  cultures  of  B.  typhosus,  Bact.  pestis,  and  Msp. 
comma  would  cause  precipitates  when  mixed  with  the  blood  serum  taken 
from  cases  of  these  diseases.  The  precipitin  reaction  is  definite  and 
specific.  The  protein  substance  used  in  immunization  or  concerned 
in  the  infection  is  the  only  one  which  is  precipitated  when  the  anti- 
serum  is  added.  To  the  protein  substance  which  produces  the  precipi- 
tins  the  name  precipitincgen  is  applied.  To  that  substance  in  the  blood 
serum  and  body  fluids  of  the  immunized  or  infected  animal  or  person 
the  name  precipitin  is  applied.  The  combination  between  the  precipi- 
tinogen  and  the  precipitin  forms  a  new  chemical  substance  known  as  a 
precipitate.  Precipitin  may  be  formed  in  various  parts  of  the  body, 
for  example,  in  the  parenchymatous  cells  of  the  organs  and  by  the 
leucocytes.  Bact.  diphtheria  will  not  act  as  a  precipitinogen  and 
will  not  produce  precipitins.  This  is  practically  the  only  bacterium 
which  will  not  yield  these  antibodies. 

Normal  Precipitins. — Precipitins  for  alien  blood  serums  have  been 
found  in  the  organs  and  blood  of  seemingly  normal  animals.  Normal 
precipitins  for  bacterial  proteins  have  not  been  demonstrated  to  a 
certainty. 

Mechanism  of  the  Formation  of  Precipitins. — The  mechanism 
of  the  formation  of  precipitins  is  similar  to  that  of  other  antibodies. 
When  the  precipitinogen  is  injected  into  the  body  of  an  animal,  it 
combines  with  certain  of  the  body  cells,  occupying  chemical  receptors 
which  otherwise  would  be  used  for  the  taking  up  of  food  products. 
As  a  result  the  cells  produce  new  receptors  and  the  number  of  these 
more  than  compensate  for  the  ones  already  utilized.  The  chemical 
receptors  are  finally  thrown  off  into  the  body  fluids  and  form  the  pre- 


IMMUNITY   AND   SUSCEPTIBILITY  719 

cipitins.  It  is  supposed  that  the  precipitinogen  contains  haptophore 
receptors  which  combine  with  the  haptophile  receptors  of  the  cells. 
When  these  haptophile  receptors  are  regenerated  and  produced  in 
excess,  as  before  stated,  they  are  thrown  off  into  the  body  fluids  and  are 
really  what  we  know  as  precipitins.  Precipitins  are  produced  most 
commonly  for  widely  different  or  heterologous  substances  or  sera 
(heteroprecipitins) . 

Auto  precipitins  and  Isoprecipitins. — It  has  been  demonstrated 
that  animals  will  not  produce  precipitins  for  their  own  protein  sub- 
stances. For  example,  if  an  animal  is  bled  and  injected  with  its  own 
blood  serum  an  antibody  will  not  be  produced.  Therefore,  autopre- 
cipitins  do  not  occur.  Again  it  has  been  shown  that  only  in  rare 
instances  do  animals  produce  precipitins  for  members  of  the  same  spe- 
cies. For  example,  if  an  animal,  such  as  a  goat,  is  bled  and  the  blood 
serum  injected  into  another  goat,  it  is  only  in  rare  cases  that  the  second 
goat  will  produce  an  antibody  which  is  capable  of  producing  precipi- 
tation of  the  proteins  in  the  first  goat's  blood  serum.  Such  precipitins 
are  known  as  isoprecipitins  and  occur  only  in  a  very  small  per  cent  of 
cases  and  with  no  regularity. 

The  Phenomena  of  Specific  Inhibition. — When  precipitins  are  heated 
to  low  temperature  (50°  to  60°)  or  are  subjected  to  the  action  of 
light  or  certain  chemicals,  their  power  to  produce  a  precipitate  when 
combined  with  a  precipitinogen  is  destroyed.  The  precipitin  which 
has  been  heated  becomes  a  precipitoid  similar  to  an  agglutinoid  or  a 
toxoid.  Their  ability  to  combine  with  the  precipitinogen  still  remains. 
It  is  possible,  therefore,  for  precipitoids  to  combine  with  all  the  avail- 
able precipitinogen  so  that  when  fresh  precipitin  is  added  no  precipitate 
will  occur.  This  is  known  as  specific  inhibition  and  sometimes  leads 
to  very  confusing  findings  in  the  study  of  these  immune  bodies. 

Antiprecipitins. — When  an  animal  has  produced  a  precipitin  in  its 
blood  serum  due  to  the  injection  of  the  antigenous  substance  which  in 
this  instance  is  known  as  the  precipitinogen,  this  precipitin,  which  is 
a  definite  antibody,  may  be  used  for  the  immunization  of  another 
animal  and  an  antiprecipitin  produced;  that  is,  a  body  which  will 
combine  with  the  precipitin  in  such  a  way  as  to  prevent  precipitation 
when  this  substance  is  combined  with  the  precipitinogen.  This 
is  then;  in  fact,  an  antiantibody  and  is  practically  the  only  example  we 


72O   MICROBIOLOGY   OF  DISEASES   OF   MAN  AND   DOMESTIC   ANIMALS 

have  in  immune  reactions  of  such  a  substance.  The  antiantibody  is 
the  limit  for  antibody  formation. 

The  Precipitinogen. — As  before  stated,  the  precipitinogen  is  any 
protein  substance  which  will  cause  the  formation  of  precipitins.  Cer- 
tain of  the  precipitinogens  are  composed  of  two  groups,  one  which  is 
thermostable  and  another  which  is  thermolabile.  Therefore,  when 
these  precipitinogens  are  heated  and  this  thermolabile  substance  de- 
stroyed there  results  a  substance  which  is  exactly  similar  to  the  precipi- 
toid  produced  by  heating  the  precipitin.  Such  bodies  are  known  as 
the  precipitoids  of  the  precipitinogen  in  distinction  from  the  precipitoids 
of  the  precipitin.  These  precipitoids  retain  their  power  to  combine  with 
precipitin,  but  no  precipitate  results  on  such  combination. 

The  Precipitate. — When  precipitin  and  precipitinogen  combine  it 
requires  some  little  time  before  precipitation  occurs.  This  is  dependent 
upon  the  temperature  (37°  best)  and  certain  other  factors.  The 
presence  of  the  trace  of  organic  acids  materially  facilitates  this  reaction. 
Furthermore,  the  reaction  will  not  take  place  without  the  presence  of 
certain  electrolytes  or  salts. 

Coprecipitins. — The  phenomena  of  " group  precipitation"  does  not 
occur  as  often  as  does  "  group  agglutination."  The  bacterial  precipitins 
are  very  markedly  specific  but  some  of  the  blood  precipitins  are  not 
so  specific.  For  example,  in  a  case  where  two  rabbits  have  been  im- 
munized, one  with  the  blood  serum  of  man  and  the  other  with  the  blood 
serum  of  the  monkey,  it  is  found  that  the  serum  of  the  rabbit  immunized 
to  human  blood  serum  will  precipitate  monkey  blood  serum  to  a  less 
degree,  of  course,  than  human  serum.  This  is  due  to  the  fact  that  there 
are  certain  chemical  substances  in  common  in  the  blood  sera  of  the 
monkey  and  man.  There  are  other  rare  instances  of  coprecipitins 
which  will  not  be  discussed. 

The  Forensic  Use  of  Precipitins. — The  precipitins  are  of  use  on  ac- 
count of  their  great  specificity  in  the  identification  of  various  albumi- 
nous substances.  They  have  been  used,  for  example,  in  the  identifica- 
tion of  bloods.  Before  the  knowledge  of  the  precipitins  was  available, 
the  only  means  of  determining  one  blood  from  another  was  by  means  of 
the  microscopic  examination  of  the  corpuscles.  If  the  corpuscles  were 
in  a  good  condition,  it  was  possible,  for  example,  to  differentiate  between 
a  mammalian  and  fowl  blood,  on  account  of  the  nucleation  of  the  cor- 
puscles of  the  latter.  By  the  use  of  the  spectroscope  it  was  also  possible 


IMMUNITY   AND    SUSCEPTIBILITY  721 

to  determine  whether  a  particular  stain  was  blood  or  not.  When  it 
came  to  determining  the  exact  species  from  which  the  blood  came  it 
was  impossible.  By  means  of  the  precipitins  this  can  be  done.  For 
example,  a  stain  which  is  supposed  to  be  blood  is  carefully  dissolved 
out  in  0.85  per  cent  sodium  chloride  solution  and  placed  in  a  sterile 
test-tube.  A  series  of  animals,  such  as  rabbits,  have  been  immunized 
to  the  various  known  blood  sera  and  after  immunization  their  sera  are 
drawn  off.  These  sera  contain  the  precipitins  for  the  various  sera  and 
corpuscles  used  in  immunization.  These  precipitins  are  combined 
separately  in  small  test-tubes  with  the  salt  solution  preparation  of  the 
blood  in  question.  A  precipitate  occurs  when  the  corresponding  pre- 
cipitating serum  is  added.  It  is  necessary,  of  course,  to  place  these 
preparations  in  the  incubator  at  37°.  By  this  method  all  types  of 
mammalian  blood  may  be  separated  from  each  other  with  the  possible 
exception,  as  before  stated,  of  monkey  and  human  blood.  In  this 
instance  it  is  necessary  to  make  careful  comparisons  in  order  to  deter- 
mine the  concentration  of  the  precipitins.  The  precipitins  may  also 
be  used  in  the  identification  of  various  meats  and  other  albuminous 
substances  such  as  eggs. 

In  some  ways  the  precipitins  resemble  colloids  and  it  has  been  shown 
that  organic  colloidal  substances  such  as  ferric  hydroxide,  etc.,  when  in 
aqueous  solution,  may  be  precipitated  by  the  addition  of  certain  elec- 
trolytic salts.  The  precipitation  occurs  in  this  instance  in  a  very 
similar  manner  to  that  of  the  organic  precipitins. 

THE  THEORIES  OF  IMMUNITY 

Various  theories  have  been  proposed  which  attempt  to  account  for 
the  resistance  naturally  present  in  animals,  and  the  resistance  which 
may  be  artificially  produced.  One  of  the  first  theories  proposed  was 
the  so-called  noxious  retention  theory  which  held  the  view  that  in  natural 
immunity  there  were  natural  noxious  substances  present  in  the  body 
which  prevented  the  growth  of  the  infectious  microorganisms.  In 
acquired  immunity  it  was  supposed  that,  as  the  result  of  an  infection, 
specific  noxious  substances  were  produced  and  consequently  new  infect- 
ing microorganisms  of  the  same  species  as  those  producing  the  original 
infection  were  unable  to  grow.  This  theory  has  long  been  discarded. 
Another  theory,  for  a  time  prominent,  was  known  as  the  exhaustion 
theory.  It  was  conceived  that  natural  immunity  was  due  to  the  fact 

46 


722    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

that  the  body  tissues  did  not  possess  the  necessary  food  products  for 
the  invading  microorganisms  and  that  in  acquired  immunity  these 
necessary  food  products  were  exhausted  completely  so  that  when  a  sec- 
ond infection  was  attempted  none  could  possibly  occur.  This  theory 
has  also  been  discarded. 

One  of  the  most  prominent  theories  is  the  one  which  has  been 
held  more  recently,  with  some  modifications,  namely,  the  chemical 
side-chain  theory  of  Ehrlich.  It  is  claimed  that  tissue  cells  are  made 
up  of  definite  chemical  substances  which  possess  chemical  side-chains 
which  are  open  for  chemical  combination  with  other  substances. 
It  is  by  means  of  these  chemical  side-chains  that  food  products  are 
absorbed  and  assimilated  by  the  cells.  Furthermore,  it  is  by  means 
of  these  chemical  side-chains  that  toxins  and  various  poisons  are 
absorbed  by  the  cells.  It  seems  to  have  been  clearly  demonstrated 
that  as  a  result  of  the  absorption  by  certain  cells  of  the  body  of  toxic 
substances,  particularly  bacterial  toxins,  that  the  cells  are  stimulated 
and  produce  or  open  up  a  excenss  of  these  chemical  side-chains  for 
combination  with  various  substances.  It  is  conceived  that  if  enough 
toxin  (not  enough  to  kill  the  cells),  is  assimilated  by  the  cells  the 
chemical  side-chains  which  are  definite  chemical  substances  will  be 
split  off  from  the  original  cell  compound  and  escape  into  the  circulation. 
It  is  these  escaped  chemical  side-chains  which  constitute  the  antitoxin 
or  bactericidal  substances.  In  the  case  of  antitoxins,  they  possess 
a  maximum  affinity  for  the  toxin  and  will  combine  with  the  toxin  much 
more  readily  than  the  toxin  will  combine  with  the  remaining  chemical 
side-chains  of  the  original  cell  compound.  In  the  case  of  bacteri- 
cidal substances  they  will  combine  with  the  bacteria  and  destroy  them 
and  liberate  in  this  way  the  endotoxins  which  may  subsequently  combine 
with  an tiendo toxin  (?)  or  tissue  cells.  Inasmuch  as  no  antiendo- 
toxins  are  ever  produced,  the  presence  of  bactericidal  substances  in 
a  large  percent  of  instances  is  a  detrimental  factor.  The  production 
of  antiendotoxins  by  some  method  or  other  is  extremely  desirable. 
Since  the  majority  of  our  diseases  are  due  to  bacteria  producing  endo- 
toxins, such  a  product  would  be  of  immense  value  in  combating  these 
infections.  The  chemical  theory  of  Ehrlich  explains  many  features 
of  the  phenomena  of  immunity.  This  theory  has  been  the  basis  of 
nearly  all  of  the  preceding  discussions  on  the  various  antibodies. 


IMMUNITY   AND    SUSCEPTIBILITY  723 

Metchnikoff  suggested  what  may  be  called  a  phagocytic  theory 
of  immunity.  According  to  his  ideas  and  those  belonging  to  his 
school,  the  phagocytes,  and  principally  the  mononuclear  and  poly- 
morphonuclear  leucocytes,  are  concerned  in  immunity.  He  explains 
natural  immunity  to  toxins  on  the  basis  of  an  increased  toxin-absorp- 
tive power  on  the  part  of  these  cells  for  toxins.  He  explains  natural 
antibacterial  immunity  as  an  increased  power  of  phagocytosis  for  the 
invading  microorganism  by  the  leucocytes.  He  conceived  that  in 
acquired  immunity  to  toxins  these  cells  develop  as  the  result  of  an  in- 
fection or  artificial  injection  of  microorganisms,  an  increased  power  of 
absorption  of  toxin  and  the  power  of  producing  antitoxin,  and  that 
acquired  immunity  to  bacteria  producing  endotoxins  is  due  to  the  in- 
creased power  of  the  phagocytes  to  ingest  and  digest  invading  micro- 
organisms. 

We  find  the  best  explanation  for  the  phenomena  of  immunity  in 
both  the  theories  of  Ehrlich  and  Metchnikoff.  Undoubtedly  certain 
forms  or  types  of  immunity  are  due  to  definite  chemical  substances 
known  as  antitoxins  or  bactericidal  substances,  while  other  types  are 
due  to  the  activity  of  the  phagocytes. 


CHAPTER  III* 
MANUFACTURE  OF  VACCINES 

INTRODUCTION 

On  July  i,  1902,  by  Act  of  Congress,  the  Secretary  of  the  Treasury, 
through  the  Public  Health  Service,  was  placed  in  control  of  all  manu- 
facture and  sale  of  viruses,  sera,  toxins,  and  analogous  products  for 
human  use.  In  order  to  manufacture  and  place  such  products  upon 
the  interstate  market,  any  individual  or  corporation  must  secure  a 
license  from  the  Secretary  of  the  Treasury  through  the  Surgeon  General 
of  the  United  States  Public  Health  Service.  All  candidates,  before 
securing  federal  approval  for  such  licenses,  must  allow  federal  inspectors 
the  privilege  of  examining  their  laboratories,  including  the  details 
involved  in  the  processes  of  manufacture.  At  frequent  intervals  the 
Hygienic  Laboratory  purchases  samples  of  licensed  products  upon  the 
open  market  for  the  purpose  of  subjecting  these  to  careful  examination. 
If  the  samples  of  any  products  are  found  to  be  misrepresented  as  to 
potency  or  kind  and  amount  of  preservative,  or  if  contaminating  organ- 
isms are  present,  the  manufacturer  is  immediately  notified  to  recall 
such  products  from  the  market. 

July  i,  1913,  by  a  similar  Congressional  Act,  the  Secretary  of  Agri- 
culture, through  the  Bureau  of  Animal  Industry,  was  authorized  to 
regulate  the  preparation  and  sale  of  viruses,  sera,  toxins  and 
analogous  products  intended  for  use  in  the  treatment  of  domestic 
animals. 

The  federal  control  of  the  manufacture  of  vaccines,  sera,  toxins 
and  other  biologic  products  related  to  specific  infectious  diseases,  has 
reduced  to  a  minimum  the  danger  formerly  involved  in  the  use  of  such 
materials. 

For  one  who  is  not  a  student  of  microbiology  and  preventive 
medicine,  or  not  familiar  with  the  technic  involved  in  preparing  biologic 
materials  such  as  sera,  tuberculins  and  vaccines  it  is  difficult  to 

*Prepared  by  W.  E.  King. 

724 


MANUFACTURE    OF  VACCINES  725 

realize  the  various  steps  necessary  in  the  production  of  a  safe  and  active 
product.  The  manipulations  attending  the  preparation  of  the  materials 
require  large  equipment,  expensive  apparatus  and  the  services  of  trained 
laboratory  experts.  Animals  which  are  used  in  the  work  must  be 
quarantined  and  carefully  inspected  before  being  placed  under  treat- 
ment. The  sanitary  conditions  of  the  laboratories,  operation  rooms 
and  stables  must  be  of  the  best. 

Infection  of  the  animal  organism  is  due  to  absence  of  natural  or 
acquired  resistance.  The  natural  resistant  forces  of  the  animal  body 
may  be  such  that  insusceptibility  to  specific  microbial  invasion  is 
present;  such  a  condition  is  called  natural  immunity.  Acquired  im- 
munity, on  the  other  hand,  refers  to  a  condition  in  which  the  natural 
susceptibility  of  the  animal  body  is  replaced  by  a  temporary  or 
permanent  resistance  toward  specific  microbial  invasions.  Acquired 
immunity  may  be  active  or  passive,  and  may  be  brought  about  by  appli- 
cation of  a  vaccine  or  an  antiserum.  The  application  of  smallpox 
vaccine  causes  a  specific  reaction  in  the  body,  stimulating  the  develop- 
ment of  natural  defences  against  smallpox  virus,  and  is  followed  by  a 
condition  of  active  immunity  which  is  relatively  permanent  in  dura- 
tion. The  use  of  diphtheria  antitoxin,  which  contains  the  antibodies 
capable  of  neutralizing  the  diphtheria  toxin  molecules,  results  in  passive 
immunity  and  affords  temporary  protection. 

ACTIVELY  IMMUNIZING  SUBSTANCES  (VACCINES) 

Vaccines*  are  essentially  weakened  or  modified  viruses.  Such 
materials  as  blackleg  and  anthrax  vaccines  may  be  used  with  safety, 
as  a  rule,  only  on  animals  which  are  free  from  the  specific  disease 
in  question,  because,  theoretically,  if  a  specific  vaccine  were  applied  to 
a  patient  suffering  from  a  given  infectious  disease,  the  introduction  of 
the  attenuated  organisms,  or  virus,  would  tend  to  augment  the 
virulence  of  the  infection.  The  action  of  such  vaccines  is  preventive 
or  prophylactic,  and  not  curative. 

ATTENUATED  VIRUSES. — There  are  several  methods  which  may  be 
employed  in  attenuating  or  modifying  viruses.  The  processes  involve 
the  treatment  of  viruses  in  such  ways  that  they  may  be  injected  into 
the  normal  animal  body  without  danger  of  producing  serious  symptoms 

*  The  term  "vaccine"  is  also  loosely  applied  to  bacterins,  bacterial  vaccines  or  suspensions 
containing  killed  microorganisms. 


726   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

or  lesions,  while  at  the  same  time  sufficient  specific,  infectious  qualities 
must  be  present  to  produce  mild  reactions.  The  successful  vaccine 
is  attenuated  or  modified  to  a  degree  which  insures  both  safety  and 
activity.  The  following  are  the  more  important  methods  used  to 
modify  viruses : 

Attenuation  by  growth  at  a  temperature  above  the  optimum.  This  is 
illustrated  by  Pasteur's  method  of  preparing  anthrax  vaccine. 

Attenuation  by  passage  of  the  virus  through  some  species  other  than 
the  animal  for  which  the  virus  is  specific.  Smallpox  vaccine  may  be 
regarded  as  a  virus  modified  by  passage  through  a  heifer  or  other 
animal. 

Attenuation  of  the  virus  by  drying  at  constant  temperature.  The 
Pasteur  method  of  prophylactic  treatment  for  rabies  is  based  upon  this 
method. 

Attenuation  by  chemicals.  The  growth  of  certain  pathogenic 
bacteria  in  the  presence  of  weak  antiseptics  reduces  their  disease- 
producing  activities. 

Other  methods  of  modifying  viruses  for  the  purpose  of  active  im- 
munization: 

The  simultaneous  method  or  hypodermic  application  of  the  virus 
together  with  protective  serum,  as  in  hog  cholera  vaccination. 

The  association  or  combination  of  the  specific  pathogenic  bacteria 
with  those  of  other  species  as  illustrated  by  the  apparent  restraining 
action  of  yeasts  upon  pyogenic  bacteria  and  the  antagonism  which 
Ps.  pyocyanea  exerts  toward  Bact.  anthracis. 

The  filtration  of  liquid  cultures  of  pathogenic  organisms,  such  as 
Bact.  diphtheria  or  B.  tetani,  and  the  consequent  separation  of  the 
organisms  from  the  toxin.  The  toxin  is  used  to  immunize  animals  in 
the  production  of  antitoxin. 

The  destruction  of  young  living  cultures  of  specific  bacteria  by 
moist  heat  at  a  temperature  slightly  above  their  thermal  death-point. 
Heated  cultures  of  B.  typhosus  and  Bact.  pestis  are  used  as  prophylactics 
against  typhoid  fever  and  bubonic  plague. 

There  are  many  vaccines  in  practical  and  experimental  use  at  the 
present  time.  Among  those  which  are  of  recognized  value  as  shown  by 
extensive  practical  use  and  reliable  clinical  statistics,  the  following 
are  the  most  important:  smallpox  vaccine,  blackleg  vaccine, rabies 
vaccine,  typhoid  vaccine  and  perhaps  Pasteur's  anthrax  vaccine. 


MANUFACTURE    OF  VACCINES  727 

The  simultaneous  method,  or  injection  of  hyperimmune  serum,  to- 
gether with  the  specific  virus  is  used  in  vaccinating  against  hog- 
cholera,  cattle  plague  (Rinderpest),  anthrax  and  foot-and-mouth 
disease.  Asiatic  cholera,  bubonic  plague,  tuberculosis,  acne,  pertussis, 
pneumonia,  canine  distemper,  furunculosis,  septicaemia  hemorrhagica, 
gonorrhoea  and  various  inflammatory  processes  are  treated,  practically 
and  experimentally,  by  various  methods  of  vaccination,  either  as 
prophylactic  or  curative  measures. 

SMALLPOX  VACCINE. — The  first  experiments  relative  to  vaccination 
against  smallpox  date  back  to  1796.  Prior  to  that  time,  the  only 
specific  preventive  method  used  in  warding  off  this  disease  depended 
upon  the  inoculation  of  healthy  individuals  with  smallpox  virus  from 
a  mild  case  of  the  disease.  The  present  method  of  vaccination  util- 
izes cowpox  virus  as  the  protective  material.  It  has  not  been  con- 
clusively determined  that  cowpox  in  cattle  and  smallpox  in  man 
possess  intimately  related  causative  factors,  but  notwithstanding, 
abundant  evidence  proves  the  efficacy  of  cowpox  virus  as  a  specific 
prophylactic  against  smallpox  in  man. 

In  the  practical  preparation  of  smallpox  vaccine,  the  virus  or 
"seed"  is  first  secured  by  removing  the  pulp  from  the  vesicles  which 
appear  on  infected  heifers.  Most  laboratories  which  engage  in  this 
work  use  a  stock  mixture  of  cowpox  virus  which  originated  from 
spontaneous  cases  of  cowpox,  and  which  is  known  to  produce  active 
smallpox  vaccine. 

Great  care  is  exercised  in  the  selection  and  preparation  of  animals 
used  in  making  the  vaccine.  Heifers  (calves  or  yearlings)  are  most 
frequently  used  in  this  work,  older  cattle  being  employed  in  a  few 
European  laboratories.  When  first  purchased  these  animals  are 
placed  in  a  detention  stable  where  they  are  inspected  by  a  qualified 
veterinarian  and  carefully  tested  for  tuberculosis.  If,  after  several 
weeks'  quarantine,  they  are  passed  as  healthy  in  every  way,  they 
are  admitted  to  the  vaccine  laboratory  after  their  bodies  have  been 
scrubbed  with  soap  and  water  and  a  weak  antiseptic  solution. 

The  operating  room  and  propagating  ward  should  be  constructed 
with  a  view  to  thorough  cleanliness.  Concrete  floors,  enameled  walls 
and  ceilings  and  simple  sanitary  apparatus  should  characterize  the 
appointments.  Floors,  walls,  ceilings  and  all  equipment  of  these  rooms 


728   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

should  be  carefully  cleansed  with  disinfectant  solutions  at  frequent 
intervals. 

After  the  heifers  are  prepared  for  the  work,  they  are  inoculated  with 
the  seed  virus.  The  animal  under  treatment  is  placed  on  a  special 
operating  table,  the  ventral  surface  of  the  body  is  shaved  and  cleansed, 
and,  with  a  sterile  instrument,  is  scarified  in  parallel  straight  lines  over 
the  greater  portion  of  the  abdomen  and  inner  surface  of  the  flanks. 
The  stock  virus  or  "seed"  is  inoculated  in  the  scarified  areas  and  the 
animal  is  released  and  placed  in  the  propagating  room.  During  the 
process  of  propagation  of  the  vaccine  all  possible  precautionary  meas- 
ures should  be  used  to  avoid  the  introduction  of  contaminating  bacteria. 
It  is  important  that  an  attendant  be  constantly  present,  day  and  night, 
whose  duty  it  is  to  remove  instantly  all  dirt  and  faeces  and  keep  the 
room  as  clean  and  free  from  microbial  contamination  as  possible. 

At  the  expiration  of  from  seven  to  nine  days,  characteristic  vesicles 
will  have  developed  on  the  inoculated  areas.  These  are  filled  with  a 
thick,  sticky,  purulent  material.  At  this  time  the  animal  is  removed  to 
the  operating  table,  the  field  of  operation  is  washed  with  sterile  water 
and  the  contents  of  the  vesicles  removed  with  a  sterile  curette.  Accord- 
ing to  regulations  of  the  Federal  Government  all  animals  used  in  this 
work  must  be  slaughtered  before  the  vaccine  is  removed  and  later 
submitted  to  careful  autopsy.  After  removing  the  pulp,  or  vaccine, 
the  material  is  handled  under  aseptic  precautions  and  mixed  with 
about  50  per  cent  glycerin,  which  serves  as  a  preservative.  Small 
portions  of  the  material  are  then  inoculated  into  guinea-pigs  for  safety 
tests  and  the  product  is  placed  in  the  refrigerator.  Under  the  in- 
fluence of  the  glycerin  extraneous  microbial  contamination  gradually 
disappears.  Potency  tests  of  the  vaccine  are  conducted  by  the  cutane- 
ous application  of  the  vaccine  on  calves,  rabbits  or  on  slightly  scarified, 
scrotal  surfaces  of  guinea-pigs.  In  addition  to  the  safety  and  potency 
tests,  inoculations  are  made  into  culture  media  which  are  placed  under 
both  aerobic  and  anaerobic  conditions  to  insure  the  absence  of  harmful 
bacteria.  For  the  detection  of  the  presence  of  B.  tetani  the  product  is 
submitted  to  a  special  test  by  transferring  i  c.c.  into  a  quantity  of 
glucose  beef  bouillon  or  other  special  culture  media,  placing  the 
culture  under  anaerobic  conditions  and  incubating  at  body-temperature 
for  about  ten  days.  After  the  incubation  any  resulting  growth  is 
removed  by  filtration  and  the  filtrate  is  injected  into  guinea-pigs. 


MANUFACTURE    OF  VACCINES  729 

The  absence  of  symptoms  in  the  treated  animals  shows  that  no  tetanus 
toxin  has  been  elaborated  in  the  culture  medium  and  therefore  that 
the  vaccine  does  not  contain  B.  tetani. 

After  the  tests  are  completed,  the  product  is  distributed  under 
aseptic  conditions,  in  small,  sterile,  capillary  tubes  sealed  in  sterile, 
glass  containers,  properly  labeled,  dated  and  kept  in  the  refrigerator 
until  placed  upon  the  market. 

If  kept  in  a  cold  dark  place,  smallpox  vaccine  retains  its  protective 
activity  for  a  considerable  period.  Under  the  influence  of  heat  and  light 
it  rapidly  deteriorates.  For  this  reason  it  is  difficult  to  ship  the  vaccine 
to  tropical  countries.  Under  suitable  conditions  the  product  should 
remain  active  for  a  period  of  about  one  year. 

BLACKLEG  VACCINE. — The  production  of  blackleg  vaccine  depends 
upon  the  use  of  a  virulent  culture  of  B.  anthracis  symptomatici  (B. 
chauvai,  B.  gangraena  emphysematosa) .  A  heifer  s  inoculated  with 
a  small  portion  of  the  virus  and  rapid,  acute  symptoms  are  usually 
produced.  Death  usually  supervenes  in  about  three  days.  The  car- 
cass and  ward  are  thoroughly  disinfected,  the  body  of  the  animal  is 
suspended,  and,  after  again  carefully  disinfecting  the  outside  of  the 
body,  portions  of  the  skin  are  removed  and  the  muscular  tissue  is  in- 
spected. Those  areas  of  the  muscles  which  show  the  dark  color,  gas- 
eous formation  and  characteristic  lesions  of  blackleg,  are  removed  to  the 
laboratory  and  examined  microscopically  for  the  presence  of  the  specific 
organisms.  After  the  muscle  is  freed  from  the  gross  connective  tissue, 
it  is  suspended  in  strips  or  finely  chopped,  and  allowed  to  dry  spon- 
taneously. It  is  then  ground  and  sterile  water  is  added  until  the  mass 
becomes  pasty  or  putty-like  in  consistency,. after  which  the  material  is 
placed  in  small  shallow  pans  and  attenuated  by  drying  at  tempera- 
ture of  85°  to  100°  for  six  or  seven  hours.  In  preparing  the  "single 
vaccine"  most  laboratories  attenuate  the  virus  by  drying  at  an  average 
temperature  of  about  90°  for  six  hours.  In  addition'  to  the  aseptic 
precautions  observed  in  conducting  the  above  processes,  microbial 
contamination  is  practically  eliminated  by  the  devitalization  and 
probable  death  of  any  extraneous  vegetative  forms  during  the  attenua- 
tion process. 

Blackleg  vaccine  (single)  is  tested,  according  to  the  method  recom- 
mended by  the  Bureau  of  Animal  Industry,  U.  S.  Dept.  of  Agriculture, 
as  follows:  A  series  of  eight  guinea  pigs  are  injected  intramuscularly 


730   MICROBIOLOGY   OF  DISEASES    OF   MAN   AND   DOMESTIC  ANIMALS 

with  the  vaccine  under  test;  three  each  with  three-fourths  the  dose  for 
cattle,  three  each  with  one-half  dose  and  the  remaining  two  with  one- 
third  dose. 

Temperatures  of  the  test  animals  should  be  recorded  for  three  sub- 
sequent days.  Vaccine  of  proper  strength  is  indicated  when  thermal 
reactions  occur  in  practically  all  the  test  animals  together  with  local 
reactions  in  some  instances.  None  of  the  animals  in  the  series  should 
die. 

As  an  additional  test  for  potency  a  heifer  may  be  injected  sub- 
cutaneously  with  one  dose  and  a  few  weeks  after  the  vaccination  the 
animal  may  be  exposed  to  the  disease  by  receiving  an  injection  of  the 
virulent  living  organisms.  If  the  animal  remains  normal  the  activity 
of  the  product  is  indicated.  In  order  to  test  the  vaccine  in  regard  to 
safety,  heifers  may  be  injected  with  several  doses  each.  The  absence  of 
severe  disturbances  shows  that  the  material  may  be  used  without 
danger. 

For  the  purpose  of  eliminating  possible  danger  from  the  use  of 
blackleg  vaccine  a  "double  vaccine"  may  be  employed.  This  consists 
of  two  vaccines,  each  possessing  different  degrees  of  attenuation,  which 
are  controlled  by  the  degree  of  heat  and  the  period  of  time  used  in  attenu- 
ating the  organisms  in  the  affected  muscle  tissue.  When  the  final 
product,  either  single  or  double  blackleg  vaccine,  is  ready  for  use  it  is 
usually  distributed  in  the  form  of  a  powder,  prepared  threads  or  small 
pills.  The  latter,  first  suggested  by  Hough  ton  in  1898,  are  injected 
hypodermically. 

Blackleg  Aggressin. — Blackleg  Aggressin  is  a  tissue  extract  con- 
taining the  immunity-producing  substances  which  are  naturally  present 
in  the  tissue  of  calves,  dead  from  acute  blackleg.  The  tissue  juices 
of  calves  dead  from  the  disease  as  a  result  of  inoculation  with  pure 
strains  of  blackleg  bacillus  are  recovered  from  the  affected  tissues 
and  rendered  free  from  the  blackleg  organism  and  extraneous  contami- 
nation by  filtration.  The  dose  for  cattle  is  5  c.c. 

Blackleg  Aggressin  may  be  tested  for  activity  or  potency  by  the 
injection  of  a  series  of  guinea-pigs  as  follows :  Two  pigs  each  are  injected 
with  i,  2,  and  3  c.c.  of  the  aggressin  under  test.  After  ten  days  the 
series  of  pigs  together  with  two  controls  are  injected  with  twice  the 
minimum  lethal  dose  of  virulent  blackleg  culture  or  virus.  The  con- 
trol pigs  should  die  in  the  usual  time  and  at  least  four  of  the  series  of 


MANUFACTURE    OF   VACCINES  731 

six  vaccinated  pigs  should  live.  Blackleg  Aggressin  may  also  be  sub- 
jected to  potency  test  by  injection  of  calves  with  5  c.c.  each,  or  with 
varying  doses.  If  prepared  according  to  standard  methods  from  care- 
fully selected  strains  of  blackleg  bacillus,  the  calves  receiving  dosage 
of  3  to  5  c.c.  should  live,  following  the  inoculation  of  the  animals  with 
virulent  blackleg  virus,  ten  days  after  receiving  the  aggressin. 

Blackleg  Filtrate. — Blackleg  nitrate  is  a  cultural  product  con- 
taining the  metabolic  substances  resulting  from  the  growth  of  the 
blackleg  bacillus  in  liquid  culture  media.  After  incubation,  usually 
about  seven  days,  or  until  optimum  growth  has  taken  place  and  the 
cultures  are  checked  for  purity,  a  preservative  is  added  and  the  organ- 
isms are  removed  by  passage  through  Berkefeld  niters.  The  method 
of  testing  for  potency  is  similar  to  that  for  blackleg  aggressin. 

RABIES  VACCINE. — The  successful  preventive  treatment  for  rabies, 
or  hydrophobia,  resulted  from  the  brilliant  researches  of  Pasteur. 
The  method  devised  by  Pasteur  in  1885,  with  some  modifications,  con- 
tinues to  be  the  only  practical,  specific  preventive  treatment  for  rabies. 
This  treatment  consists  of  a  series  of  vaccinations,  each  vaccination 
involving  the  application  of  rabies  virus  having  a  known  degree  of 
attenuation.  In  each  succeeding  application  of  modified  rabies  virus 
the  patient  receives  increasingly  more  virulent  material  until  finally 
active  immunity  is  acquired  and  subsequent  attack  from  the  disease 
is  successfully  resisted. 

The  preparation  of  rabies  vaccine  begins  with  the  attenuation  of  a 
virus  having  a  known  degree  of  virulence.  The  material  may  be  secured 
from  an  ordinary  case  of  "street  rabies."  A  dog  suffering  from  the 
disease  is  killed  and  a  small  portion  of  the  brain  removed.  The  brain 
tissue  is  emulsified  in  sterile  water  or  salt  solution  and  a  few  drops  of  the 
material  thus  suspended  in  liquid,  are  injected  subdurally  into  a  rabbit. 
This  may  easily  be  accomplished  by  trephining  the  skull,  after  anaesthe- 
tizing the  animal,  and  with  a  small  syringe  inoculating  a  few  drops  of 
the  suspension  just  under  the  exposed  dura  mater.  The  inoculation  of 
ordinary  rabies  virus  usually  produces  symptoms  of  "dumb  rabies " 
and  the  death  of  the  animal  in  fourteen  to  eighteen  days.  In  order  to 
increase  the  virulent  properties  of  the  same  strain  of  rabid  material,  it 
is  transmitted  from  rabbit  to  rabbit  by  subdural  inoculations  until 
the  incubation  period  is  shortened  to  about  six  days.  Experience  has 
shown  that  when  the  virus  has  reached  its  maximum  degree  of  virulence 


732    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

for  the  rabbit,  the  animal  shows  symptoms  on  the  sixth  or  seventh 
day  after  inoculation.  When  the  virus  attains  this  degree  of  virulence 
it  is  called  "fixed  virus"  and  may  be  used  in  the  preparation  of  the 
vaccine.  The  " fixed  virus"  or  spinal  cord  of  the  rabbit  which  has 
succumbed  to  the  disease  in  six  or  seven  days,  is  removed  aseptically 
and  placed  in  a  special  drying  chamber.  The  cords  are  suspended  over 
caustic  potash  and  dried  at  a  temperature  of  23°  for  a  period  of  from 
one  to  ten  or  fifteen  days. 

The  treatment  of  the  patient  consists  in  the  hypodermic  applica- 
tion of  the  "fixed  virus"  which  has  been  attenuated  by  drying.  The 
exact  nature  of  the  vaccine  used  in  the  initial  vaccination  and  the  time 
consumed  in  the  series  of  injections  depend,  to  some  extent,  upon  the 
case  in  hand.  Frequently,  the  patient  is  first  vaccinated  with  a  sus- 
pension of  a  spinal  cord  which  has  been  attenuated  by  drying  for  four- 
teen or  fifteen  days.  On  the  succeeding  days  of  the  treatment  use  is 
made  of  the  suspension  of  spinal  cords,  which  have  been  less  and  less 
attenuated.  The  treatment  usually  lasts  about  twenty  days  or  until 
the  patient  has  received  an  injection  of  the  least  attenuated  "fixed 
virus." 

It  is  very  important,  when  one  is  bitten  by  a  rabid  animal,  that  the 
Pasteur  treatment  be  begun  as  early  as  possible,  in  order  that  active 
immunity  may  be  secured  before  the  expiration  of  the  incubation  period. 
In  many  of  the  larger  cities  of  the  United  States,  for  some  time,  labora- 
tories have  been  maintained  for  the  purpose  of  administering  the 
Pasteur  treatment.  More  recently,  commercial  laboratories  have 
developed  methods  of  preparation  and  distribution,  so  that  any  phy- 
sician may  purchase  the  vaccine  and  administer  it  to  his  patients. 

Hogyes*  substituted  dilutions  of  the  "fixed  virus"  for  the  dried 
spinal  cords.  For  the  initial  treatment,  a  few  cubic  centimeters  of  a 
i :  10,000  dilution  were  used.  In  the  succeeding  injections  graduated 
dilutions  were  employed.  While  the  work  of  Hogyes  has  been  con- 
firmed by  other  investigators,  the  method  is  not  generally  regarded 
as  possessing  the  safety  of  the  original  Pasteur  treatment. 

Harris  f  has  devised  a  simple  method  of  preparing  the  vaccine  by 
freezing  the  infected  spinal  cord  of  the  rabbit  with  C0%  snow,  and  then 
drying  the  material  in  vacuo  over  sulphuric  acid  at  a  temperature  of 
10°  to  15°. 

*  Hogyes,  Acad.  des  Sciences  de  Budapest,  Oct.  17,  1897. 
t  Harris,  Jour.  Infect.  Dis.,  1912,  10,  p.  369. 


MANUFACTURE    OF  VACCINES  733 

The  product  is  kept  in  the  refrigerator  in  hermetically  sealed  vials. 
It  is  claimed  that  the  material  so  prepared  maintains  its  original 
strength  or  infectivity  several  months. 

Cummings'*  method  consists  in  the  dialysis  of  the  rabic  material 
in  standard  suspensions.  Dialysis  for  twelve  to  twenty-four  hours 
possesses  the  advantage  of  destroying  the  infectivity  of  the  virus, 
without  disturbing  its  immunizing  properties. 

DORSET- NILES  (HOG-CHOLERA)  SERUM. f — To  prepare  the  material 
for  this  process  of  immunization  it  is  first  necessary  to  secure  a  virulent 
strain  of  hog  cholera  virus.  This  may  be  obtained  from  any  typical 
outbreak  of  the  disease.  A  specimen  of  blood  may  be  drawn,  asep- 
tically,  from  the  carotid  artery  of  a  pig  suffering  from  the  disease,  and 
tested  for  activity.  Frequently  a  given  strain  of  virus  may  not  produce 
the  acute  form  of  hog  cholera.  In  attempting  to  raise  the  virulence  of 
a  relatively  weak  virus  it  may  be  passed  through  a  series  of  young  pigs 
until  it  uniformly  produces  symptoms  after  four  to  six  days'  incubation 
and  death  in  less  than  fifteen  days.  None  except  a  virus  having  this 
degree  of  activity  should  be  used  in  manufacturing  the  hyperimmune 
serum. 

The  virulent  blood  used  in  the  process  of  hyperimmunization 
should  be  obtained  from  susceptible  pigs  weighing  from  25  to  50  kg. 
(50  to  loo  pounds)  each.  The  animals  to  be  used  as  the"hyperim- 
munes"  should  be  healthy  hogs,  each  weighing  from  100  kg.  to  150  kg. 
(220  to  330  pounds)  and  possessing  either  natural  or  acquired  immunity 
to  the  disease.  The  blood  is  best  secured  from  a  diseased  pig  by 
suspending  the  animal  with  the  head  down  covered  with  a  shroud 
wet  with  a  disinfectant  solution.  The  neck  is  shaved  and  disinfected. 
A  small  incision  is  made  on  the  median  line  through  which  a  specially 
devised  bleeding  knife,  properly  sterilized,  is  introduced.  The  blade 
of  this  knife  severs  the  large  vessels  at  the  base  of  the  heart  and  the 
blood  flows  through  the  hollow  handle  into  sterile  containers.  After 
the  blood  is  obtained  it  is  defibrinated,  the  serum  separated  from  the 
clot,  and  the  clot  discarded.  The  number  of  pigs  necessary  to  furnish 
sufficient  virus  for  the  hyperimmunization  of  one  hog  depends  upon  the 
weight  of  both  the  virus  pigs  and  the  immune  hog. 

The  immune  hogs  may  be  hyperimmunized  either  by  the  "slow" 

*  Proceedings  isth  International    Congress  on  Hygiene  and  Demography,  Washington 
D.  C.,  1912. 

t  U.  S.  Bureau  of  Animal  Industry,  Bull.  No.  102. 


734   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

or  "quick"  method.  In  the  former,  now  seldom  used,  the  animals 
receive  several  injections  at  intervals  of  every  few  days,  each  succeeding 
dose  being  increased  in  proportion  to  the  weight  of  the  animal.  In  the 
"quick"  method  the  virus  is  injected  in  one  large  dose,  the  amount  being 
determined  by  the  weight  of  the  animal.  The  virus  may  be  injected 
intramuscularly,  intraperitoneally  or  intravenously,  the  latter  method 
now  being  used  almost  exclusively.  Ten  days  to  two  weeks  after  the 
hyperimmune  hog  has  received  the  last  injection  of  virus,  the  animal 
is  ready  for  bleeding.  When  bled  from  the  tail,  the  end  of  the  appen- 
dage is  severed  with  a  sharp  instrument,  several  hundred  cubic  centi- 
meters of  blood  are  collected  aseptically,  defibrinated,  a  preservative 
added  and  the  material  placed  in  the  refrigerator.  This  process  is 
repeated  several  times,  at  intervals  of  one  week  to  ten  days,  when  the 
animal  is  ready  for  final  bleeding. 

By  this  procedure  all  the  blood  is  secured  from  the  animal  according 
to  the  method  described  for  bleeding  virus  pigs.  The  "slaughter" 
method,  used  in  many  laboratories,  consists  of  only  the  final  bleeding, 
thus  eliminating  tail  bleedings.  As  a  rule  the  different  lots  of  serum 
representing  the  different  bleedings  from  several  hyperimmune  hogs 
are  mixed  and  the  whole  subjected  to  test.  In  order  to  test  the 
potency  of  the  product  eight  susceptible  pigs,  each  weighing  about  23 
kg-  (50  pounds)  are  inoculated  subcutaneously,  each  with  2  c.c.  of 
virus.  Six  of  these  pigs  are  simultaneously  injected  with  graduated 
doses  (15  to  25  c.c.)  of  the  serum  under  test.  If  the  hyperimmune 
serum  possesses  potency  the  test  pigs  should  remain  in  a  normal  con- 
dition throughout  the  test,  except  for  the  presence  of  thermal  reactions 
and  slight  clinical  symptoms,  while  the  two  control  pigs  should  show 
severe  symptoms  in  five  or  six  days  and  should  die  in  less  than 
fifteen  days. 

The  practical  method  of  treatment  in  the  field  consists  in  the  simul- 
taneous injection  of  the  hyperimmune  serum  and  virus  (double  treat- 
ment), into  healthy  hogs  for  the  purpose  of  immunization.  The 
amount  of  hyperimmune  serum  which  should  be  injected  varies  from 
30  c.c.  to  90  c.c.,  depending  upon  the  weight  of  the  hog  to  be  treated. 
Thus,  a  hog  weighing  34  kg.  to  45  kg.  (75  to  100  pounds)  usually  receives 
40  c.c.  of  serum,  together  with  i  c.c.  of  virus.  The  usual  dose  of  virus 
for  hogs  above  34  kg.  (75  pounds)  weight  is  2  c.c.  For  pigs  weighing 
less  than  23  kg.  (50  pounds)  %  c.c.  of  virus  should  be  injected. 


MANUFACTURE    OF   VACCINES  735 

ANTHRAX  VACCINE. — While  several  methods  have  been  used  in  vac- 
cinating against  anthrax,  probably  the  most  important,  at  present, 
is  that  devised  by  Pasteur.  This  method  consists  in  the  use  of  cultures 
which  have  been  attenuated  by  growth  on  artificial  culture  media  at 
temperatures  above  the  optimum.  The  inoculation  of  such  attenuated 
cultures  into  healthy  animals  results  in  active  immunization. 

The  stock  culture  of  Bact.  anthracis  is  usually  obtained  from  the 
blood  of  a  typical  case  of  anthrax.  The  culture  is  transferred  to  agar 
or  broth  and  incubated.  Two  vaccines  are  prepared,  the  first  being 
less  active  than  the  second.  Vaccine  No.  i,  is  made  by  placing  in 
suspension  in  sterile,  physiological  salt  solution  or  other  liquid,  the 
anthrax  organisms  which  have  been  grown  at  a  temperature  of  42°  for 
a  period  of  fifteen  to  twenty  days.  Vaccine  No.  2  consists  of  a 
similarly  treated  culture  of  Bact.  anthracis  which  has  grown  at  a  tem- 
perature of  42°  for  ten  to  fifteen  days.  Tests  of  both  vaccines  for 
activity  and  safety  are  made  by  animal  inoculations.  Vaccine  No.  i 
should  kill  white  mice  but  should  not  cause  fatal  results  in  guinea-pigs 
or  rabbits.  Vaccine  No.  2  should  prove  fatal  for  both  white  mice  and 
guinea-pigs,  but  not  for  rabbits. 

Healthy  animals  are  first  injected  subcutaneously  with  about  i  c.c. 
of  vaccine  No.  i.  Several  days  or  a  few  weeks  after  the  application  of 
vaccine  No.  i,  the  second  vaccine  is  injected.  A  severe  reaction  and 
sometimes  death  follows  the  use  of  the  vaccine.  Accidents  of  this  kind 
have  resulted  from  careless  methods  employed  in  standardizing  and 
administering  the  vaccine.  The  most  important  objection  to  Pasteur's 
anthrax  vaccine  is  due  to  the  danger  involved  in  the  use  of  the 
living,  attenuated  anthrax  organisms. 

Scalvo*  advocates  the  use  of  the  serum  from  animals  actively  im- 
munized to  anthrax.  This  method  may  be  employed  either  in  the 
form  of  the  immune  serum  alone,  or  the  immune  serum  and  anthrax 
culture  simultaneously. 

Eichhorn  f  advises  the  use  of  antianthrax  serum  for  curative 
purposes,  and  the  simultaneous  treatment  with  antiserum  and  a  care- 
fully standardized  spore  vaccine  as  a  preventive.  When  vaccine  alone 
is  to  be  employed  Eichhorn  prefers  the  spore  vaccine  rather  than  the 
ordinary  Pasteur  vaccine. 

*  Scalvo,  Centralbl.  f.  Bakt.,  1899,  26,  p.  425. 
t  Eichhorn,  Bull.  No.  340,  U.  S.  Dept.  of  Agr. 


736   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

TUBERCULOSIS  VACCINE. — Among  the  experimental  products  for 
the  prevention  of  animal  tuberculosis  may  be  mentioned  von  Behring's 
"  bo vo- vaccine."  The  technique  involved  in  the  preparation  of  this 
vaccine  is  not  generally  known.  Romer*  describes  the  material  as 
being  composed  of  the  living  tubercle  organisms  which  are  dried  for  a 
period  of  thirty  days  in  sealed  glass  tubes.  After  this  process  of  attenua- 
tion the  organisms  are  injected,  in  carefully  graduated  doses,  into 
healthy  calves.  Field  tests  which  have  been  made  upon  calves  with 
bo  vo- vaccine  indicated  unsatisfactory  results. 

In  human  practice  various  tuberculins  prepared  both  from  the 
bouillon  culture  and  from  the  cellular  elements  of  Bact.  tuberculosis 
are  used  as  therapeutic  and  diagnostic  agents.  Products  containing 
the  cellular  elements  are  similar  in  nature  to  bacterial  vaccines. 

BACTERIAL  VACCINES  (BACTERINS) 

Opsonins  may  be  defined  as  the  elements  in  the  blood  or  body  fluids 
which  are  capable  of  modifying  invading  bacteria  in  such  a  way  that 
they  become  ready  prey  to  the  leucocytes.  In  the  presence  of  opsonins, 
therefore,  phagocytic  activity  is  increased.  Opsonins  are  apparently 
distinct  from  agglutinins,  lysins,  and  other  analogous  substances, 
because  different  degrees  of  heat  are  necessary  for  their  destruction. 
Moreover,  a  given  serum  may  agglutinate,  or  may  exert  lytic  action, 
without  possessing  opsonic  activity. 

Wright  and  Douglas  first  advanced  the  theory  of  opsonic  action,  and 
suggested  that  the  subcutaneous  injection  of  a  given  species  of  bacteria, 
killed  by  heating,  caused  the  blood  of  the  treated  individual  to  exert 
greater  opsonic  activity  toward  the  species  of  organisms  in  question. 
The  results  of  the  work  of  others  proved  to  be  confirmatory. 

To  prepare  a  bacterial  vaccine,  the  specific  organism  is  isolated  and 
after  being  grown  for  twenty-four  hours  or  longer  at  a  temperature  of 
37°,  it  is  emulsified  in  sterile  physiological  salt  solution,  heated  at 
approximately  60°,  or  killed  by  the  use  of  chemical  agents,  standardized 
as  to  the  number  of  bacteria  present  in  i  c.c.  of  the  emulsion,  and  a 
preservative  added. 

If  the  patient  and  attending  physician  are  conveniently  situated  in 
respect  to  a  laboratory,  the  "opsonic  index"  may  be  taken  before  and 
during  the  treatment.  This  consists  in  the  determination  of  the  aver- 

*  Romer,  Beitrage  z.  Exp.  Therapie.  1904,  J. 


MANUFACTURE    OF  VACCINES  737 

age  number  of  the  given  species  of  bacteria  ingested  by  the  leucocytes 
of  the  patient,  as  compared  to  that  which  the  leucocytes  of  normal 
blood  are  capable  of  destroying.  It  is  usually  found  that  immediately 
following  the  injection  of  specific  bacterial  vaccine  there  is  a  "  negative 
phase"  during  which  the  leucocytes  of  the  patient  destroy  a  smaller 
number  of  bacteria.  This  is  followed  by  a  "positive  phase,"  character- 
ized by  more  active  phagocytosis.  For  practical  purposes  the  determi- 
nation of  the  opsonic  index  is  unnecessary  as  the  clinical  reaction  fol- 
lowing the  injection  of  a  given  vaccine  indicates  correct  dosage  and 
progressive  results  of  the  treatment. 

The  use  of  bacterial  vaccines  has  yielded  excellent  results  espe- 
cially in  the  curative  treatment  of  furunculosis,  acne,  sycosis,  puerperal 
infection,  arthritis  and  other  affections  caused  by  pyogenic  organisms, 
and  in  chronic  infections  of  the  genito-urinary  tract.  The  material 
may  be  used  in  the  form  of  "autogenous"  or  "stock"  vaccines.  An 
autogenous  (personal)  bacterial  vaccine  is  one  prepared  from  a  culture 
of  the  specific  organism  isolated  from  the  case  under  treatment. 
Bacterial  vaccines,  prepared  from  stock  cultures  of  the  specific  organ- 
isms, may  be  manufactured  and  kept  until  needed  for  use.  Some  of 
the  more  common  stock  bacterial  vaccines  represent  the  following 
organisms  alone  or  in  various  combinations:  Strept.  pyo genes,  M. 
pyo genes  var.  (albus,  aureus  and  citreus),  M.  gonorrhoea,  Bact.  pertussis, 
M.  pneumonic?,  and  B.  coli  communis. 

The  study  of  bacterial  vaccines  occupies  a  position  of  so  much 
importance  in  preventive  medicine  and  therapeutics  that  many  new 
combinations  of  killed  bacteria  are  being  constantly  added  to  the 
list  of  experimental  products.  Some  of  these  have  been  under  ob- 
servation for  a  considerable  time  and  are  recognized  as  possessing 
valuable  properties. 

TYPHOID  FEVER. — The  typhoid  bacterial  vaccine  of  Wright*  is 
generally  accepted  as  a  valuable  preventive  against  infection.  For 
prophylactic  treatment,  a  series  of  three  hypodermic  injections 
(500,000,000,  1,000,000,000  and  1,000,000,000)  of  killed  typhoid 
organisms  are  usually  given. 

Typhoid-paratyphoid  vaccine  (Bacterin)  is  frequently  used,  con- 
sisting of  Bacillus  typhosus  1000  million,  Bacillus  paratyphosus  A  590 
million,  and  Bacillus  paratyphosus  B  500  million. 

*  Wright,  Jour,  of  Hyg.  2,  1902,  p.  385. 
47 


738   MICROBIOLOGY   OF   DISEASES   OF   MAN   AND   DOMESTIC  ANIMALS 

PNEUMONIA. — Experiences  of  recent  years,  especially  in  the  army, 
have  proved  the  value  of  pneumococcus  vaccine,  particularly  as  a 
prophylactic  agent.  The  usual  dose  consists  of  500  million  killed 
pneumococci. 

INFLUENZA-PNEUMONIA. — Among  the  various  bacterins  which 
were  prepared  and  used  experimentally  during  the  influenza-pneumonia 
epidemic  of  1918-1919,  the  preparation  suggested  by  Rosenow*  of 
the  Mayo  Foundation  has  found  ratner  extensive  use.  This  bacterin 
is  composed  of  carefully  selected  strains  of  pneumococci,  types  i,  2,  3 
and  4,  Strept.  hemolyticus  and  B.  influenza. 

CANINE  DISTEMPER. — Ferry, f  corroborated  by  Torrey  t  and  Mc- 
Gowan||  found  this  disease  to  be  primarily  an  infection  of  the  upper 
respiratory  tract  due  to  a  small  motile  bacillus,  B.  bronchisepticus. 

Ferry  and  Torrey  proved  that  suspensions  of  killed  cultures  of  this 
organism  will  immunize  dogs  against  experimental  inoculations  as  well 
as  against  the  ordinary  street  infection.  The  bacterial  vaccine  is  being 
used  for  prophylactic  purposes  in  graduated  doses  of  200,  400  and  600 
million  bacteria  per  c.c.,  given  at  intervals  of  about  five  days. 

ASIATIC  CHOLERA. — Two  methods  of  vaccination  against  this 
disease  have  been  proposed  and  statistics  which  relate  to  field  tests 
show  positive  results  with  both.  The  method  of  vaccination  resulting 
from  the  work  of  Haffkine§  depends  upon  the  use  of  cultures  of  the 
spirillum  of  Asiatic  cholera,  attenuated  by  growth  at  temperatures 
above  the  optimum.  Vaccines  of  different  strengths  are  used.  KolleU 
has  proposed  the  use  of  heated  (killed)  cultures  of  the  organism. 
Strong**  has  developed  a  vaccine  for  Asiatic  cholera  consisting  of  the 
filtrates  from  suspensions  of  killed  and  living  Msp.  comma  (Sp.  cholera 
Asiatics).  This  vaccine  is  standardized  in  terms  of  immunity  units, 
one  unit  "equaling  the  amount  of  immune  serum  which  will  protect  a 
guinea-pig  of  250  g.  weight  against  the  intraperitoneal  inoculation  of  ten 
times  the  fatal  dose  of  living  cholera  organisms." 

*  Rosenow,  E.  C.,  Jour.  A.  M.  A.,  Vol.  72,  No.  22,  p.  1604. 

Rosenow,  E.  C.  and  Sturdivant,  B.  P.,  Jour.  A.  M.  A.,  Vol.  73,  No.  6,  p.  396. 
t  Ferry,  Am.  Vet.  Rev.,  1910,  Vol.  37,  p.  499.    Jour,  of  Infec.  Dis.,  1911,  vol.  8,  p.  399. 
t  Torrey,  Jour,  of  Med.  Research,  1913,  Vol.  27,  291. 
||  McGowan,  Jour,  of  Path,  and  Bact.,  1911,  Vol.  15,  P-  372. 
§  Haffkine,  Brit.  Med.  Jour.,  1895,  2.  pp.  727,  1509. 
t  Kolle,  Deut.  med.  Woch.,  1897,  23.  p.  4. 
"*  Strong,  Philip.  Am.  Med.,  1903,  Vol.  6,  p.  272. 

Strong,  Philip.  Jour.  Sci.,  1907,  Vol.  2.  p.  155. 


MANUFACTURE   OF  VACCINES 


739 


BUBONIC  PLAGUE. — Practically  the  same  methods  of  procedure 
have  been  followed  in  the  experimental  vaccination  against  bubonic 
plague  as  in  the  case  of  Asiatic  cholera.  Cultures  of  the  plague  bacillus, 
killed  by  heating  at  a  temperature  of  60°  for  one  hour,  have  been  used 
with  success. 

SENSITIZED  VACCINE 

Besredka*  has  developed  modified  bacterial  vaccines  known  as 
sensitized  vaccines.  In  the  preparation  of  these  the  living  micro- 
organisms are  brought  into  contact  with  the  homologous  antisera 
and  the  mixtures  allowed  to  stand  for  approximately  twenty-four  hours 
at  room  temperature.  The  organisms  are  then  removed  by  centrifugal- 
ization,  washed  and  placed  in  suspension.  The  remaining  processes 
of  manufacture  are  similar  to  those  employed  in  the  preparation  of 
ordinary  bacterial  vaccines. 

Besredka  and  his  associates  explain  the  advantage  of  sensitized 
vaccines  by  the  fact  that  in  such  preparations  the  microorganisms,  by 
reason  of  having  been  in  contact  with  homologous  antisera,  are 
sensitized  with  specific  amboceptors.  Therefore,  the  sensitized  organ- 
isms are  capable  of  immediately  combining  with  complement,  when 
introduced  in  the  blood  of  the  patient,  and  prompt  immunization  should 
follow. 

Both  living  and  killed  sensitized  microorganisms  have  been  used 
experimentally,  Besredka f  having  advocated  the  use  of  the  former  as 
devoid  of  harmful  properties  and  more  certain  of  successful  results. 
Sensitized  vaccines  are  still  in  the  experimental  stage,  and  their  ad- 
vantage over  the  ordinary  bacterial  vaccines  is  at  present  a  debated 
question. 

TOXIN — ANTITOXIN  MIXTURE. 

Babes,  J  in  1895,  first  suggested  the  use  of  diphtheria  toxin  and  antitoxin  mixture 
as  a  method  of  immunization  against  diphtheria.  Through  the  work  of  Park  and 
Zingher||  and  others  who  preceded,  this  method  is  being  adopted  in  practice, 
especially  as  a  means  of  prophylaxis  against  diphtheria  in  schools  and  hospitals. 
The  mixture  consists  of  active  diphtheria  toxin  and  antidiphtheritic  serum  in  the 
proportion  of  80  per  cent,  of  the  L  4-  dose  of  toxin  to  one  unit  of  antitoxin. 

*  Besredka,  Compt.  Rend,  de  1'Acad.  Sci.,  1902,  134,  p.  1330. 
t  Besredka,  Bull,  de  1'lnst.,  Pastteur,  1910,  8,  p.  241. 
J  Babes,  Bui.  Acad.  de  Med.,  Paris,  1895,  34,  p.  216. 
||  Park  and  Zingher,  Jour.  A.M.A.  1915,  65,  p.  2214. 


CHAPTER  IV* 

THE  MANUFACTURE  OF  ANTISERA  AND   OTHER  BI- 
OLOGICAL PRODUCTS  RELATED  TO  SPECIFIC 
INFECTIOUS  DISEASES. 

The  principles  involved  in  serum  therapy  are  those  of  passive 
immunization.  Therefore,  the  employment  of  an  antiserum  as  a  pre- 
ventive or  curative  measure  is  an  attempt  to  supply  the  patient  with 
certain  specific  substances  which  are  capable  of  neutralizing  and  de- 
stroying the  specific  toxic  materials  and  pathogenic  microorganisms. 
Presumably,  the  patient  receives  nothing  in  antisera  which 
stimulate  the  development  of  protective  bodies.  Active  immunity 
does  not  follow  as  in  the  case  of  vaccine  treatment.  As  the  result 
of  serum  treatment,  the  patient  enjoys  relatively  temporary  pro- 
tection (preventive  treatment),  or  cessation  of  pathologic  processes 
(curative  treatment),  because  of  the  application  of  specific  antisub- 
stances.  The  substances  contained  in  the  serum  are  developed 
in  the  blood  of  some  other  species,  as  the  horse,  through  repeated 
injections  of  the  animal  with  the  specific  organism  in  question  or 
its  toxin. 

Antisera  are  divided  into  antitoxic  and  antimicrobial  sera.  An 
antitoxic  serum  is  one  possessing  substances  which,  in  contact  with 
the  specific  toxin,  unite  with  it,  forming  chemically  stable  and  physi- 
ologically inert  compounds.  Under  the  term  "antitoxic  serum," 
in  addition  to  antidiphtheritic  and  antitetanic  sera,  are  grouped  anti- 
sera  for  the  soluble  toxins  of  B.  botulinus  (specific  meat  poisoning), 
abrin,  ricin  and  crotin  (plant  toxins),  snake  venom  and  spider  toxin, 
and  the  soluble  toxins  of  Bact.  Welchii  (gas  gangrene)  and  of  B. 
anthracis  symptomatic*  (blackleg  in  cattle). 

The  antimicrobial  sera  constitute  the  majority  of  serum  products. 
Included  among  these  are  antimeningococcic,  antistreptococcic,  anti- 
gonococcic,  antistaphylococcic,  antityphoid,  antidysenteric,  antirabic, 

*Prepared  by  W.  E.  King. 

740 


THE   MANUFACTURE    OF   ANTISERA  741 

antipneumococcic,  antituberculosis,  antiplague,  anticholera,  antihog 
cholera,  antianthrax  sera  and  sera  for  swine  erysipelas,  fowl  cholera, 
white  scours  of  calves,  sheeppox,  foot-and-mouth  disease,  canine  dis- 
temper, rinderpest  and  spotted  fever.  The  action  of  this  group  is 
directed  more  especially  against  the  specific  microorganisms  involved, 
resulting  in  dissolution  of  the  cells  or  lysis  due  to  lytic  bodies  in  the 
antisera  (bacteriolysins). 

In  addition  to  the  presence  of  lysins  in  antimicrobial  sera,  other 
antisubstances  are  known  to  exist,  as  agglutinins,  bacteriotropins 
(opsonins),  and  precipitins.  The  antibody  content  of  antimicrobial 
sera  is  comparatively  little  understood  and  the  clinical  interpreta- 
tion of  lysins,  agglutinins  and  precipitins  is  not  clear. 

ANTITOXIC  SERA 

DIPHTHERIA  ANTITOXIN. — A  culture  of  the  organism  may  readily  be 
secured  from  the  throat  of  a  patient  by  transferring  some  of  the  diph- 
theritic exudate,  on  a  sterile  cotton  swab,  to  Loeffler's  blood-serum 
culture  medium.  After  the  growth  of  the  bacteria  at  incubator  tem- 
perature, contaminating  organisms  may  conveniently  be  eliminated  by 
the  inoculation  of  a  guinea-pig  and  the  isolation  of  the  diphtheria  organ- 
isms from  the  tissues.  A  pure  culture  is  necessary  in  the  preparation 
of  the  antitoxin,  but  any  given  culture  should  not  be  relied  upon  until 
tests  have  been  made  of  the  final  product. 

To  produce  the  diphtheria  toxin  with  which  the  antitoxin  horses  are 
treated,  the  diphtheria  organisms,  in  pure  culture,  are  transferred  to 
beef  broth,  contained  in  large  flasks,  and  incubated  at  a  temperature  of 
37°.  A  rapid  growth  takes  place,  during  which  the  specific  toxin  is 
elaborated  by  the  organisms.  After  a  period  of  incubation  of  seven 
days,  the  bouillon  culture  is  removed  from  the  incubator,  examined 
microscopically  in  order  to  make  sure  that  contamination  is  not  present, 
a  preservative  is  added,  usually  carbolic  acid,  trikresol,  or  purified  cre- 
sols,  and  the  organisms  are  removed  from  the  culture  by  passing  the 
liquid  through  a  Berkefeld  filter.  The  filtrate  (diphtheria  toxin)  is 
then  placed  in  the  refrigerator  until  used. 

The  horses  which  are  used  in  the  manufacture  of  antidiphtheritic 
serum,  as  well  as  for  the  preparation  of  other  antisera,  must  be 
submitted  to  rigid  inspection  before  being  placed  on  the  treatment. 
These  animals,  when  purchased,  are  placed  in  a  detention  stable  for 


742    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

several  weeks.  During  this  time  they  are  subjected  by  a  qualified 
veterinarian  to  a  thorough  physical  examination  and  to  the  mallein 
test  for  glanders.  Finally,  only  those  animals  which  are  pronounced 
normal  in  every  way  are  admitted  to  the  antitoxin  stables.  The  stables 
and  the  operating  rooms  with  their  appointments,  which  are  designed 
for  the  antitoxin  horses,  should  be  constructed  with  a  view  to  perfect 
sanitation  and  cleanliness.  Concrete  floors,  sanitary  stalls,  mangers, 
stocks  and  apparatus,  good  water,  free  ventilation  and  plenty  of  light 
should  characterize  the  quarters. 

The  antitoxin  horses  are  injected  subcutaneously  with  the  diphtheria 
toxin.  The  initial  dose  of  toxin  usually  consists  of  only  a  fraction  of  a 
cubic  centimeter,  then  increasingly  larger  doses  are  administered  until 
the  animals  are  finally  able  to  receive  300  c.c.  or  more  at  a  single  treat- 
ment. The  intervals  between  injections  and  the  rate  of  increase  of 
succeeding  doses  at  any  given  time  depend  upon  the  condition  of  the 
animal.  During  this  treatment  a  constant  process  of  antitoxin  forma- 
tion is  taking  place  in  the  body  of  the  horse.  In  order  to  produce  a  po- 
tent serum,  the  injection  of  the  toxin  should  be  continued  throughout 
the  course  of  treatment  as  rapidly  as  the  resulting  reactions,  following 
each  injection  of  the  animal,  will  allow. 

After  the  completion  of  the  initial  toxin  treatment,  which  occupies 
a  period  of  from  six  weeks  to  three  months,  the  horse  is  allowed  a 
rest  of  about  two  weeks,  during  which  time  all  the  toxin  which  has  been 
injected  should  be  absorbed.  During  the  remainder  of  the  antitoxin- 
producing  period  of  the  animal's  life  (approximately  two  years),  treat- 
ment with  diphtheria  toxin  is  continued  at  regular  intervals.  When 
desired,  a  small  sample  of  blood  serum  may  be  secured  from  the  horse  for 
preliminary  potency  tests.  Finally,  the  animal  is  bled  from  the 
jugular  vein,  under  aseptic  conditions.  As  much  blood  is  secured  as 
the  horse  can  conveniently  yield,  varying  in  quantity  from  10  to  15  1. 
The  blood  may  be  drawn  through  a  sterile  canula  and  rubber  tube  into 
tall,  sterile  glass  cylinders.  After  the  blood  has  clotted  the  serum 
separates  and  at  the  end  of  twenty-four  to  forty-eight  hours,  the  clear, 
amber-colored  fluid  is  poured  from  the  cylinders  into  large,  sterile  glass 
containers,  a  preservative  is  added  and  the  material  is  transferred  to 
the  laboratory.  The  serum  is  then  passed  through  a  Berkefeld  filter. 

Each  lot  of  antidiphtheritic  serum  is  submitted  to  rigid  tests 
relative  to  potency,  safety  and  microbial  contamination.  In  deter- 


THE    MANUFACTURE    OF    ANTISERA  743 

mining  the  potency,  varying  amounts  of  the  serum  under  test  are 
mixed  with  the  L+  dose  of  diphtheria  toxin  and  injected  into  a  series 
of  guinea-pigs,  each  weighing  250  g.  *  The  L-f  dose  of  toxin  is  the  least 
amount  of  toxin,  which,  when  mixed  with  one  unit  of  standard  antitoxin 
(supplied  by  the  Hygienic  Laboratory)  and  injected  into  a  guinea-pig 
of  250  g.  weight,  is  sufficient  to  kill  the  animal  in  four  days.  From  the 
results  of  this  test  it  is  possible  to  determine  the  smallest  amount  of  the 
antitoxin  which  will  protect  a  guinea-pig  of  250  g.  weight,  when  the 
animal  has  received  simultaneously  the  L-f-  dose  of  toxin.  This  mini- 
mum amount  of  antitoxin  represents  one  unit.  Thus,  if  J^QO  c-c-  of  the 
given  antitoxin  represents  the  smallest  amount  which  is  capable  of 
neutralizing  the  L+  dose  of  toxin,  then  the  antitoxin  would  possess  a 
potency  of  500  units  per  c.c. 

In  order  that  the  antitoxin  may  be  tested  for  safety,  each  of  several 
guinea-pigs  is  injected  subcutaneously  with  about  2  c.c.  of  the  serum. 
These  animals  are  not  released  until  the  observer  is  satisfied  that  the 
serum  contains  no  injurious  properties.  For  the  purpose  of  detection 
of  microbial  contamination,  relatively  large  amounts  of  the  antitoxin 
are  placed  in  culture  media  and  incubated  under  both  aerobic  and 
anaerobic  conditions. 

Diphtheria  antitoxin  is  usually  distributed  in  glass  syringe  containers 
ready  for  immediate  use.  After  the  product  has  been  tested  relative 
to  potency,  safety  and  microbial  contamination,  it  is  put  up  in  sterile 
glass  cylinders.  These  cylinders  are  so  constructed  that  accompanying 
sterilized  needles  and  pistons  may  be  conveniently  applied  and  the  anti- 
toxin injected  hypodermically  directly  from  the  containers.  Each 
container  must  bear  a  label  indicating  the  number  of  antitoxin  units 
enclosed  and  the  date  of  preparation. 

Finally,  after  the  diphtheria  antitoxin  has  been  distributed  in  the 
glass  cylinders,  sealed  and  packed  ready  for  use,  sample  packages  are 
opened  and  examined  for  contamination,  usually  by  two  microbiologists. 
The  product  is  not  approved  until  the  independent  results  of  these  final 
tests  are  compared,  and  it  is  assured  that  microbial  contamination  is 
absent. 

All  antitoxic  sera  should  be  kept  away  from  the  light  and  at  a 
temperature  of  10°  to  15°  whenever  possible,  as  the  presence  of  heat 
and  light  causes  gradual  deterioration.  Usually  an  expiration 

*  See  Bulletin  No.  21,  Hygienic  Laboratory,  Washington,  D.  C. 


744   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

date  of  from  eighteen  months  to  two  years  is  applied  to  diphtheria 
antitoxin. 

It  has  been  demonstrated  that  the  antitoxic  content  of  serum  is 
closely  associated  with  the  globulins.  Advantage  is  taken  of  this 
fact  by  most  laboratories  in  reducing  the  volume  of  antitoxin,  or 
concentrating  the  product,  by  precipitating  the  globulins  with 
ammonium  sulphate,  redissolving  the  precipitate  and  dialyzing. 
The  concentration  of  serum  by  this  method  increases  the  unit  value 
per  volume  and  tends  to  decrease  the  occurrence  of  undesirable  second- 
ary effects  ("serum  sickness"). 

TETANUS  ANTITOXIN. — The  processes  involved  in  the  preparation 
of  antitetanic  serum  differ  but  little  from  those  employed  in  the 
manufacture  of  diphtheria  antitoxin.  The  pure  culture  of  B.  tetani 
is  inoculated  into  large  flasks  of  glucose  bouillon,  placed  under  an- 
aerobic conditions  and  incubated  at  body  temperature.  A  convenient 
method  of  excluding  free  oxygen,  in  the  presence  of  which  the  tetanus 
organisms  will  not  multiply,  consists  in  boiling  the  glucose  bouillon 
before  the  inoculation,  to  drive  off  the  oxygen,  then  covering  the 
liquid  medium  by  a  layer  of  oil.  These  cultures  are  subjected  to  a 
temperature  of  37°  for  about  two  weeks,  after  which  they  are  examined 
microscopically,  preservative  is  added  and  the  organisms  are  removed 
by  filtration.  On  account  of  the  presence  of  spores  and  the  danger  at- 
tending the  contamination  of  any  materials  or  biological  products  with 
the  tetanus  bacillus,  it  is  important  that  great  care  should  be  exercised 
in  the  nitration  and  preparation  of  the  tetanus  toxin.  Therefore,  the 
filtration  process  is  best  accomplished  in  an  isolated  room  which  is 
used  only  for  the  preparation  of  tetanus  toxin. 

Tetanus  antitoxin  is  produced  by  the  injection  of  horses  with  the 
specific  toxin  and  the  same  general  methods  and  precautions  are  ob- 
served as  in  the  preparation  of  diphtheria  antitoxin.  The  anti- 
tetanic  serum  is  tested  relative  to  potency,  safety  and  freedom  from 
microbial  contamination.  The  standard  unit  of  tetanus  antitoxin  is 
regarded  as  ten  times  the  least  quantity  of  antitetanic  serum  necessary 
to  save  the  life  of  a  3$o-g.  guinea-pig  for  ninety-six  hours,  against  the 
official  dose  of  a  standard  toxin  furnished  by  the  Hygienic  Laboratory 
of  the  Public  Health  Service. 

Tetanus  antitoxin  is  put  up  for  use  in  the  same  manner  as  diph- 

*  See  U.  S.  Treasury  Department,  Public  Health  Reports,  Vol.  XXIV,  No.  20,  1904. 


THE   MANUFACTURE    OF   ANTISERA  745 

theria  antitoxin,  being  usually  distributed  in  glass  syringe  containers. 
The  product  is  used  in  both  human  and  veterinary  practice. 

PERFRINGENS  ANTITOXIN. — During  the  last  two  years  of  the 
recent  world  war,  considerable  attention  was  devoted  to  the  experi- 
mental development  of  perfringens  antitoxin  or  anti-  gas  gangrene 
serum.  The  use  of  tbis  serum  in  conjunction  with  tetanus  antitoxin 
was  adopted  by  the  War  Department  during  the  later  months  of  the 
war.  It  is  prepared  by  the  injection  of  horses  with  Bad.  Welchii  toxin. 
The  strength  of  the  toxin  is  determined  by  the  injection  of  pigeons 
which  are  uniformly  susceptible  to  the  substance.  The  antitoxin  is 
standardized  by  injection  of  pigeons  with  mixtures  of  serum  under  test 
and  varying  amounts  of  standardized  toxin.  The  sudden  termination 
of  the  war  did  not  permit  the  accumulation  of  conclusive  evidence 
regarding  the  efficacy  of  perfringens  antitoxin. 

ANTIMICROBIAL  SERA 

In  addition  to  diphtheria  and  tetanus  antitoxins,  certain  other 
antisera  are  rapidly  attaining  practical  significance.  At  present, 
however,  no  methods  are  in  use  by  which  any  antisera  other  than, 
diphtheria  and  tetanus  antitoxins  can  be  accurately  standardized  as 
to  potency.  Nevertheless,  most  of  the  products  can  be  submitted  to 
rigid  tests  in  order  to  determine  the  presence  of  protective  qualities. 

ANTIMENINGOCOCCIC  SERUM. — Horses  are  immunized  to  cultures  of 
a  number  of  strains  of  M .  intracellularis  var.  meningitidis,  the  activity 
of  the  resulting  serum  being  determined  by  agglutination  and  com- 
plement fixation  tests.  Antimeningococcic  serum  is  used  in  the  active 
treatment  of  cerebrospinal  meningitis  and  is  administered  by  lumbar 
puncture.  The  dose  depends  principally  upon  the  age  of  the  patient 
and  the  condition  of  the  blood  pressure. 

ANTISTREPTOCOCCIC  SERUM. — Bouillon  cultures  of  Strept.  pyo genes 
are  killed  by  heating,  and  injected  into  horses  in  increasingly  larger 
doses.  Frequently,  the  killed  cultures  used  in  treating  the  horses  are 
composed  of  several  different  strains  of  the  streptococcus.  In  this  case 
the  resulting  antistreptococcic  serum  is  designated  as  "polyvalent," 
while  the  serum  obtained  after  the  injection  of  cultures  consisting  of 
but  one  strain  of  the  organism,  is  called  "  monovalent"  antistreptococcic 
serum. 


746   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

In  procuring  the  serum,  handling,  filtering,  preserving  and  dis- 
tributing for  use,  the  methods  are  practically  the  same  as  those  em- 
ployed in  the  preparation  of  antidiphtheritic  serum. 

Antistreptococcic  serum  is  carefully  tested  in  regard  to  safety  and 
freedom  from  microbial  contamination.  There  are  no  methods  avail- 
able for  definitely  standardizing  the  product.  The  serum  is  often 
efficacious  in  cases  of  strep tococcic  infection. 

ANTIGONOCOCCIC  SERUM. — Killed  cultures  of  M.  gonorrhoea  are 
injected  intraperitoneally  or  intravenously  into  large,  healthy  rams,  or 
other  animals.  The  dosage  is  increased  and  finally  live  cultures  are 
applied,  the  degree  of  immunity  acquired  being  determined  by  com- 
plement fixation  and  agglutination  tests  of  the  sera  from  the  animals. 

ANTIPNEUMOCOCCIC  SERUM. — This  is  prepared  by  the  injection  of 
horses  with  pneumococci,  type  i,  and  has  been  found  to  possess  thera- 
peutic properties  when  the  disease  is  due  to  Diplococcus  pneumonia, 
type  i.  Antipneumococcus  serum  prepared  from  types  2  and  3  has 
been  found  to  be  of  little  value  as  compared  with  serum  representing 
pneumococcus  type  i.  In  using  antipneumococcic  serum  from  type  i 
organism,  it  is  therefore  desirable,  whenever  possible,  to  isolate  and 
type  the  pneumococci  present  in  a  given  case  under  treatment. 

DORSET-NILES  (ANTI-HOG  CHOLERA)  SERUM  (HYPERIMMUNE 
SERUM)*. — This  product  has  been  described  in  the  preceding  chapter 
under  "hog  cholera  vaccine"  (Double  Treatment).  When  the  hyper- 
immune  serum  is  used  unaccompanied  by  the  virus,  either  among 
healthy  or  diseased  swine,  the  process  is  known  as  the  "Serum- Alone 
Method."  Reichelf  has  succeeded  in  producing  an  antihog  cholera 
serum  which  is  sterile  and  free  from  inert  solid  matter  by  precipitat- 
ing the  globulins.  Dorset  and  Henley  t  have  announced  the  produc- 
tion of  a  clear  and  sterile  serum  by  employing  an  extract  of  common 
garden  beans  together  with  salt  to  agglutinate  the  blood  corpuscles. 
ANTIRABIC  SERUM. — Animals  which  have  been  immunized  to  rabies 
are  bled  and  the  immune  serum  may  be  used  as  a  preventive  and 
therapeutic  agent.  While  this  product  is  not  often  employed  in  prac- 
tice, yet  it  has  been  shown  by  various  investigators  that  considerable 
protection  is  obtained  from  its  use. 

*  See  U.  S.  Bureau  of  Animal  Industry,  Bull.  No.  102. 
tReichel,  Proc.  i8th  Ann.  Mtg.  U.S.L.S.S.  Asso.,  1915,  P-  127. 
$  Dorset  and  Henley,  Jour.  Agr.  Research,  Vol.  6,  May  29,  1916. 


THE    MANUFACTURE    OF    ANTISERA  747 

ANTIDYSENTERIC  SERUM. — Experimental  monovalent  and  poly- 
valent antisera  for  epidemic  dysentery  have  been  developed  by 
Shiga  and  Flexner,  by  the  injection  of  horses  with  the  filtrates  from 
bouillon  cultures  of  the  dysentery  bacillus. 

THE  PRESERVATION  OF  ANTISERA. — The  question  of  a  proper  pre- 
servative for  antisera  has  received  much  attention.  The  problem 
of  preservation  involves  several  conditions,  as  the  ideal  preserva- 
tive, when  incorporated  in  the  proper  volume  of  serum  in  efficient 
dilutions,  must  possess  marked  inhibitive  and  germicidal  power,  it 
must  prove  inert  when  injected  into  the  patient,  and  it  must  produce 
no  objectionable  precipitation  of  serum  proteins.  At  present,  trikresol 
or  purified  cresols  (0.4  of  i  per  cent)  is  generally  employed. 

BIOLOGICAL  PRODUCTS  OTHER  THAN  VACCINES  AND  ANTISERA 

TUBERCULINS. — Koch's  Tuberculin  (Old). — Koch's  tuberculin  is  the 
concentrated;  glycerinated,  beef  bouillon  in  which  Bact.  tuberculosis  has 
been  grown.  The  active  substance  of  the  tuberculin  is  apparently  an 
albuminous  body  insoluble  in  alcohol.  The  product  is  harmless  for  the 
non-infected,  but  exerts  a  toxic  action  upon  tuberculous  individuals,  the 
reaction  being  characterized  by  a  rise  in  temperature  which  begins  two 
to  ten  hours  after  treatment,  continues  for  a  few  hours  and  finally 
subsides.  Tuberculin  (old)  is  used  as  a  diagnostic  agent  in  both  human 
and  veterinary  practice. 

Tuberculin  (old)  is  prepared  from  cultures  of  the  human  or  bovine 
variety  of  Bact.  tuberculosis.  Apparently  the  active  product  can  be 
obtained  from  attenuated  as  well  as  from  virulent  cultures.  The 
organism  is  inoculated  into  beef  bouillon  to  which  5  per  cent  glycerin 
has  been  added.  The  culture  medium  is  usually  distributed  in  flasks 
and  the  tubercle  organisms,  when  inoculated,  are  carefully  placed  on  the 
surface  of  the  medium.  The  cultures  are  incubated  at  a  temperature  of 
37°  to  38°  for  six  to  ten  weeks,  during  which  time  a  heavy  growth  slowly 
spreads  over  the  surface  of  the  medium  and  finally  falls  to  the  bottom 
of  the  flasks.  In  the  successful  preparation  of  tuberculin  it  is  important 
that  the  cultures  should  remain  undisturbed,  having  access  to  plenty 
of  air,  that  the  incubator  temperature  should  be  constantly  maintained 
without  fluctuations,  and  that  the  organisms  should  be  allowed  to  grow 
until  they  have  completely  elaborated  the  active  "tuberculinic"  sub- 
stance. After  the  growth  is  complete,  the  cultures  are  removed  from 


748   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND   DOMESTIC  ANIMALS 

the  incubator  and  sterilized  in  streaming  steam.  The  killed  cultures, 
are  then  evaporated  over  a  water  bath  to  one-tenth  the  original  volume, 
the  bacteria  are  removed  by  passing  the  cultures  through  paper  and 
Berkefeld  filter  and  a  preservative  is  added.  For  cattle  the  dose  of 
tuberculin  concentrated  by  evaporation  to  one-tenth  the  original  vol- 
ume, is  0.25  c.c.  to  0.7  c.c.  Because  of  the  fact  that  the  material  is 
thick  and  syrupy  in  consistency  and  the  dose  is  inconveniently  small, 
it  is  usually  diluted  with  seven  parts  of  weak  carbolic  acid  solution. 
During  the  preparation  it  may  be  evaporated  to  four-fifths  the  original 
volume  and  preserved  by  the  addition  of  i  per  cent,  carbolic  acid  of 
sufficient  volume  to  dilute  properly.  The  United  States  Department 
of  Agriculture,  Bureau  of  Animal  Industry,  requires  that  a  dose  of 
tuberculin  for  the  subcutaneous  test  of  cattle  shall  not  be  less  than  the 
equivalent  of  0.5  g.  of  Koch's  Old  Tuberculin.  According  to  this 
requirement  the  dosage  for  cattle  must  depend  upon  the  amount  of 
Koch's  Old  Tuberculin  contained  in  any  given  brand  of  the  product. 
The  dose  of  tuberculin  prepared  by  most  laboratories  should  not  be 
less  than  5  c.c.  as  each  c.c.  usually  contains  the  equivalent  of  o.i  g. 
Koch's  Old  Tuberculin.  The  product  should  be  tested  for  activity 
by  injecting  known  tuberculous  animals  with  the  tuberculin  under  test. 
The  presence  of  typical  reactions  in  tuberculous  animals  indicates  the 
reliability  of  the  product.  Tuberculin  may  be  subjected  to  experi- 
mental test  for  activity  by  the  method  suggested  by  the  Bureau  of 
Animal  Industry,  the  essentials  of  which  are  as  follows: 

A  series  of  guinea-pigs  are  inoculated  with  a  suspension  of  fresh 
tubercular  material  taken  from  lesions  in  tubercular  cattle.  After 
the  development  of  tuberculosis  in  the  guinea-pigs  a  second  series  of 
guinea-pigs  may  be  injected  with  a  suspension  of  tubercles  from  the 
first  series  of  infected  guinea-pigs.  These  animals  should  be  weighed 
at  regular  intervals  and  carefully  examined  for  evidences  of  tubercu- 
losis. After  a  period  usually  of  about  three  or  four  weeks,  a  series  of 
guinea-pigs  should  be  selected  which  show  unmistakable  evidence  of 
tuberculosis.  This  series  together  with  controls  may  be  injected  with 
varying  doses  of  tuberculin  under  test,  in  order  to  determine  the  degree 
of  sensitiveness  to  the  lot  of  tuberculin  in  question.  One  group  of 
tubercular  guinea-pigs  should  be  injected  with  Bureau  of  Animal  In- 
dustry Tuberculin  of  known  potency  which  serves  as  a  standard.  The 
dose  of  tuberculin  to  be  injected  in  the  test  guinea-pigs  should  be  the 


THE    MANUFACTURE    OF   ANTISERA  749 

equivalent  of  0.25  g.  of  Koch's  Old  Tuberculin  per  500  g.  weight  guinea- 

pig- 

Conclusions  may  be  drawn  from  such  tests  according  to  the  follow- 
ing suggestions  made  by  the  Bureau  of  Animal  Industry: 

If  the  tuberculin  which  is  provisionally  taken  as  the  standard  kills 
not  less  than  two-thirds  of  the  sensitized  guinea-pigs  injected  with  it 
before  the  lapse  of  twenty-four  hours,  and  the  two  normal  guinea-pigs 
injected  with  it  remain  free  from  symptoms  of  disease  excepting  the 
rapidly  passing  distress  which  may  immediately  follow  the  injection, 
it  is  required  that  any  other  sample  of  tuberculin,  if  it  possesses  a  re- 
liable degree  of  potency,  should  kill,  within  twenty-four  hours,  at  least 
half  the  sensitized  guinea-pigs  injected  with  it,  and  that  the  normal 
guinea-pigs  injected  with  it  should  be  alive  and  well  at  the  end  of 
twenty-four  hours. 

In  human,  as  well  as  in  veterinary  practice,  tuberculin  may  be 
applied  as  a  diagnostic  agent  in  various  ways.  In  addition  to  the  hypo- 
dermic injection  of  tuberculin  (old),  as  described  above,  the  method  of 
Calmette,*  von  Pirquetf  and  MoroJ  may  be  used  in  human  practice. 
Calmette's  ophthalmo  test  consists  in  the  instillation  in  the  eye  of 
Koch's  purified  or  refined  tuberculin.  Purified  tuberculin  is  prepared 
by  treating  the  original  tuberculin  with  absolute  alcohol,  washing  and 
drying  the  precipitate.  One  drop  of  a  i  per  cent  solution  of  purified 
tuberculin  is  placed  in  the  eye.  A  positive  reaction  is  manifested  by  a 
congestion  of  the  palpebral  and  ocular  conjunctiva  a  few  hours  after  the 
application  of  the  tuberculin.  The  method  of  von  Pirquet*  depends 
upon  the  cutaneous  application  of  the  tuberculin.  One  drop  of  tuber- 
culin (old)  is  placed  on  the  arm,  after  cleansing  the  skin,  and  the  small 
area  under  the  drop  is  scarified.  Two  or  more  small  areas  may  be 
treated  in  this  way,  as  well  as  a  control  area  treated  with  sterile  salt 
solution  or  a  solution  of  glycerin  and  dilute  carbolic  acid  in  substitu- 
tion for  the  tuberculin.  The  appearance  of  a  reddish  zone  in  from 
twelve  to  twenty-four  hours  indicates  a  positive  reaction.  This  area 
of  inflammation  gradually  increases  somewhat  in  elevation  and  diameter 
and  finally  subsides  in  a  few  days.  Moro's  modification  of  von  Pir- 
quet's  method  consists  in  the  use  of  tuberculin  ointment  prepared  by  the 
combination  of  tuberculin  (old)  and  anhydrous  lanolin  in  equal  parts. 

*  Calmette,  Presse  Medicale,  1907,  15. 
t  von  Pirquet,  Berl.  klin.  Woch.,  1907,  44. 
j  Moro,  Mfinch.  med.  Wch.,  1908. 


75O   MICROBIOLOGY   OF   DISEASES    OF   MAN  AND   DOMESTIC   ANIMALS 

The  ointment  is  vigorously  rubbed  on  a  small  portion  of  the  skin  of  the 
abdomen.  A  positive  reaction  is  evidenced  by  the  appearance  of  a 
distinct  granular  or  papular  eruption  at  the  point  of  application  after 
about  twenty-four  hours. 

For  the  diagnosis  of  tuberculosis  in  cattle,  the  intradermal  test  is 
generally  regarded  as  next  in  importance  to  the  older  subcutaneous  test. 
In  conducting  this  test  o.i  to  0.3  c.c.  of  a  50  per  cent  solution  of  tuber- 
culin is  injected  into  the  cuticle  layer  of  the  skin  at  the  base  of  the 
tail.  A  positive  reaction  is  present  when,  twenty-four  to  seventy-two 
hours  after  the  injection  of  tuberculin,  the  localized  area  of  skin  shows 
a  circumscribed  cedematous  swelling. 

Tuberculin  (old)  is  usually  distributed  in  small  vials,  sealed  and 
labeled.  The  labels  should  indicate  the  amount  and  dosage  and  the 
date  of  preparation.  Under  the  influence  of  light  and  heat  the  fluid 
product  may  slowly  deteriorate;  therefore,  when  possible,  it  should  be 
kept  in  the  refrigerator  until  needed. 

Other  Tuberculins.— Koch  introduced  tuberculin  "T.  R."  (tuber- 
culin residuum)  in  1897  and  tuberculin  "B.  E."  (bacillary  emulsion)  in 
1901.  The  former  is  prepared  by  repeatedly  centrifugalizing  a  suspen- 
sion of  the  dried  and  ground  tubercle  organisms  in  water.  The  super- 
natant fluid  "T.  O."  after  the  first  centrifugalization  is  discarded  and 
the  final  product  consists  of  the  constituents  of  the  bacteria  which  are 
insoluble  in  water.  One  cubic  centimeter  of  the  tuberculin  "T.  R." 
should  contain  the  equivalent  of  2  mg.  of  the  dry  tubercle  solids. 
Tuberculin  B.  E.  is  composed  of  a  suspension  of  crushed  or  thoroughly 
ground  tubercle  organisms  in  50  per  cent  glycerin  solution.  Each 
cubic  centimeter  should  contain  the  equivalent  of  5  mg.  of  tubercle 
solids.  Tuberculin  T.  R.  and  tuberculin  B.  E.  are  used  as  therapeutic 
agents,  the  latter  probably  being  regarded  with  more  favor  by  clini- 
cians. The  material  is  administered  by  subcutaneous  injection,  the 
time  intervening  between  successive  treatments  varying  from  three  to 
ten  days.  The  initial  dose  recommended  by  most  investigators, 
is  o.oooi  mg.  or  less. 

MALLEIN. — Mallein  is  prepared  from  cultures  of  Bact.  mallei  by 
practically  the  same  methods  as  those  employed  in  manufacturing 
tuberculin  from  Bact.  tuberculosis.  The  product  is  used  for  the  diag- 
nosis of  glanders.  A  few  hours  after  mallein  is  injected,  subcutaneously, 
into  glandered  horses  a  severe  local  reaction  and  a  rise  of  temperature 


THE   MANUFACTURE    OF    ANTISERA  751 

usually  follow.  The  thermal  reaction  is  very  similar  to  that  produced 
in  tuberculous  animals  by  the  injection  of  tuberculin.  The  local  swell- 
ing caused  by  mallein  treatment  is  considered  by  some  to  be  quite  as 
diagnostic  as  the  temperature  reaction. 

The  ophthalmic  mallein  test,  a  comparatively  recent  method,  which 
was  first  used  by  Choromansky,  appears  to  be  attaining  considerable 
recognition  as  a  valuable  aid  in  diagnosis.  The  test  consists  in  the 
application  of  concentrated  mallein  to  the  inner  canthus  of  the  eye. 
A  drop  of  the  concentrated  mallein  in  liquid  form  or  a  small  bit  of  the 
same  in  desiccated  condition  may  be  used.  In  a  positive  case, 
hyperemia  and  swelling  of  the  conjunctiva  and  a  purulent  exudate  at 
the  inner  canthus  of  the  eye  will  appear  from  four  to  six  hours  after  the 
instillation  of  the  mallein. 

Goodall*  advocates  the  use  of  the  intrapalpebral  mallein  test  which 
involves  the  injection  of  a  small  dose  of  mallein  under  the  skin  of  the 
eyelid. 

The  stock  culture  of  the  glanders  organism  used  in  the  preparation 
of  mallein  should  be  one  which  possesses  known  virulent  properties. 
It  is  grown  at  a  temperature  of  37°  for  three  months  in  flasks  of  glycerin 
bouillon  having  a  chemical  reaction  of  about  three  points  acid  to 
phenolphthalein.  When  the  cultures  are  removed  from  the  incubator 
they  are  heated  in  streaming  steam,  passed  through  a  Berkefeld  filter 
and  the  filtrate  is  concentrated,  preserved  and  distributed  in  labeled 
vials. 

SUSPENSIONS  FOR  THE  AGGLUTINATION  TESTS 

Agglutinins  are  hypothetical  bodies  existing  in  the  blood  and  possi- 
bly other  body  tissues,  of  an  individual  affected  with,  or  convales- 
cent from,  a  specific  infectious  disease.  The  bodies  possess  the  power 
of  "clumping"  and  precipitating  the  specific  bacteria  which  are  the 
cause  of  the  disease  in  question.  Thus,  if  a  dilution  of  blood  serum  from 
a  typhoid  fever  patient  is  mixed  with  living  typhoid  organisms,  the 
specific  agglutinins  present  in  the  serum  will  cause  the  organisms  to 
cease  their  motion  and  agglutinate  or  clump  together  in  irregular 
masses.  Normal  human  blood  serum  placed  under  the  same  conditions 
will  fail  to  cause  the  agglutination  of  the  organisms  in  similar  dilutions. 
The  agglutination  reaction  may,  therefore,  be  used  in  the  diagnosis  of 

*  Goodall,  Jour.  Comp.  Path,  and  Therap.,  1915,  Vol.  28,  p.  281. 


752    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND    DOMESTIC  ANIMALS 

certain  specific  infectious  diseases.  The  serum  must  be  properly 
diluted  in  order  that  the  reaction  may  be  of  diagnostic  value,  because 
undiluted,  normal  serum  will  cause  a  positive  agglutination  reaction 
in  most  cases. 

The  agglutination  test  is  used  as  a  practical  aid  chiefly  in  typhoid 
fever  in  man  and  glanders  in  horses.  The  test  may  be  conducted  either 
microscopically  or  macroscopically.  In  the  microscopic  method,  the 
diluted  serum  from  the  suspected  case  is  placed  under  the  microscope 
with  the  live,  specific  organisms  in  hanging  drop.  In  the  macroscopic 
method,  the  serum  is  added  to  an  emulsion  of  the  killed  (heated) 
bacteria  in  small  test-tubes,  and  the  resulting  reaction  detected  with  the 
naked  eye. 

The  emulsion,  suspension  or  "test  fluid"  for  the  typhoid  agglutina- 
tion test  is  prepared  from  a  pure  culture  of  B.  typhosus.  The  organism 
is  grown  for  twenty-four  hours  upon  agar  at  a  temperature  of  37°. 
The  growth  is  then  removed  from  the  surface  of  the  agar,  placed  in 
sterile,  physiologic  salt  solution  and  the  organisms  killed  by  heating  on 
a  water  bath  at  a  temperature  of  60°  for  one-half  hour.  The  emulsion 
is  then  roughly  standardized  by  adding  sufficient  sterile,  physiologic 
salt  solution  to  impart  to  the  fluid  the  required  degree  of  cloudiness, 
when  compared  with  control  emulsions.  To  the  suspension  of  dead 
typhoid  organisms  or  "test  fluid"  a  preservative,  usually  formalin, 
is  added  and  the  product  is-  distributed  in  properly  labeled  bottles.  In 
conducting  the  test,  the  suspected  typhoid  serum  is  placed  in  small 
tubes,  each  containing  i  c.c.  of  the  suspension  fluid,  in  such  propor- 
tions that  the  serum  is  diluted  i :  50,  i :  100,  and  i :  200.  A  flocculent 
precipitate  of  the  dead  organisms  indicates  a  positive  reaction. 

Suspension  fluid  for  the  glanders  agglutination  test  is  prepared  in 
practically  the  same  manner  as  the  typhoid  test  fluid.  The  glanders 
organisms  are  grown  on  acid  agar  and  the  suspension  fluid  is  usually 
preserved  by  the  addition  of  carbolic  acid.  In  conducting  the  glanders 
agglutination  test,  the  suspected  serum  is  usually  placed  in  the  follow- 
ing dilutions:  1:200,  1:500,  1:800,  1:1200,  and  1:1800. 

The  agglutination  reaction  has  been  applied  experimentally  and 
practically,  with  more  or  less  success,  in  the  diagnosis  of  Malta  fever, 
Asiatic  cholera,  bubonic  plague,  pneumonia,  tuberculosis,  contagious 
abortion  (bovine)  and  other  infectious  diseases. 


THE   MANUFACTURE    OF   ANTISERA  753 

SUBSTANCES  USED  FOR  DIAGNOSTIC  TESTS 

LUETIN. — Noguchi*  has  developed  a  preparation  known  as  luetin 
which  is  used  in  the  diagnosis  of  syphilis.  The  material  is  prepared 
from  a  number  of  strains  of  Spirochata  pallida  grown,  under  anaerobic 
conditions,  on  special  ascites  agar  and  bouillon  media.  After  abundant 
growth  of  the  spirochetes  occurs,  the  agar  cultures  are  ground  and  mixed 
into  a  paste.  To  this  material  fluid  cultures  are  added  in  sufficient 
proportion  to  form  a  liquid  emulsion.  The  organisms  are  then  killed 
by  heating  at  60°  for  one  hour  and  a  preservative  is  added. 

In  applying  this  diagnostic  material  to  a  suspected  syphilitic  case 
0.05  c.c.  is  very  carefully  injected  into,  not  beneath,  the  skin.  An  area 
on  the  anterp-internal  surface  of  the  upper  arm  is  usually  chosen  as  the 
s.ite  of  injection.  A  positive  diagnosis  of  syphilis  is  indicated  if,  after 
the  third  day  a  marked  cutaneous  eruption  appears  at  the  point  of 
inoculation. 

ANTIGENS. — Certain  antigens,  such  as  gonococcus  and  syphilitic 
antigen,  are  of  value  for  the  purpose  of  conducting  complement  fixation 
tests  in  laboratory  diagnosis.  Gonococcus  antigen  consists  of  an  ex- 
tract or  filtrate  prepared  from  a  suspension  of  polyvalent  gonococci. 
Syphilitic  antigen  consists  of  an  extract  prepared  from  either  luetic 
or  certain  normal  tissues  such  as  beef  or  human  heart  muscle.  Tuber- 
culosis antigen,  as  described  by  Craig, f  consists  of  the  filtrates  of 
specially  prepared  cultures  of  Bact.  tuberculosis. 

THE  ScmcK  TEST. — The  susceptibility  or  non-susceptibility  of 
individuals  to  diphtheria  may  be  determined  by  the  application  of  the 
test  described  by  Schick.J  For  this  purpose  standardized  diphtheria 
toxin  is  required.  0.1-^0.2  c.c.  of  a  relatively  fresh  normal  saline  solu- 
tion containing  J^o  minimum  lethal  dose  of  diphtheria  toxin,  for  a 
25o-g.  guinea-pig,  is  injected  intracutaneously.  The  appearance  of  a 
circumscribed  area  of  redness  at  the  site  of  injection  after  twenty-four 
to  forty-eight  hours  indicates  that  the  individual  possesses  practically 
no  immunity  against  diphtheria. 

*  Noguchi,    H. :  Jour.  Exp.  Med.,  xiv,  Vol.  16. 
t  Craig,  Am.  Jour.  Med.  Sci.,  1915.  ISO,  p.  781. 
t  Schick,  Munch.  Med.  Woch.  1913,    60,  p.  2608. 


CHAPTER  V* 

CONTROL  OF  INFECTIOUS  DISEASES 
PRINCIPLES 

That  the  infectious  diseases  can  be  controlled  depends  upon  the 
facts  that  they  arise  only  hi  the  presence  of  a  specific  living  infective 
agent;  that  they  pass  from  patient  to  prospective  patient  only  because 
the  infective  agent  passes  from  patient  to  prospective  patient;  and 
that  therefore  the  prevention  of  effective  passage  will  prevent  thje 
spread  of  the  disease.  These  preventive  measures  with  then*  natural 
incidental  developments  constitute  the  practice  of  present  public  health 
relating  to  these  diseases. 

In  general  the  infective  agent  leaves  the  body  of  the  patient  by  the 
mucus-lined  orifices  of  the  body,  the  nose  and  the  mouth,  the  anus, 
the  urethra,  the  mammae,  and  the  genital  organs.  In  general  it  must,  if 
it  is  to  infect  successfully  another  person,  reach  one  or  more  of  the  same 
mucus-lined  orifices  of  that  other  person.  Excluding  the  venereal 
diseases  the  ordinary  infectious  diseases  (tuberculosis,  typhoid  fever, 
diphtheria,  scarlet  fever,  measles,  whooping  cough,  smallpox,  chicken- 
pox,  pneumonic  plague,  leprosy)  are  received  almost  exclusively  into 
the  body  through  the  mouth  (or  nose).  While  the  passage  is  usually 
from  mucous  membrane  to  mucous  membrane  as  above  outlined,  the 
infective  agent  may  pass  effectively  from  mucous  membrane  to  cut  or 
abrased  skin  (the  uninjured  skin  is  probably  almost  always  resistant 
to  these  infections).  Again,  in  those  diseases  where  skin  lesions  are  a 
prominent  feature  (smallpox,  plague,  leprosy)  the  infective  agent  may 
pass  from  the  skin  lesions  to  a  mucous  membrane,  or  to  a  cut  or  abra- 
sion. But  these  are  rare  methods  of  transmission  as  compared  with  the 
mucous  to  mucous  forms,  except  in  syphilis  and  chancroid  where  they 
frequently  occur. 

The  routes  of  travel  between  the  patient  and  the  prospective  pa- 
tient are  many.  At  times,  mucous  membrane  may  be  applied  to 

*  Prepared  by  H.  W.  Hill. 

754 


CONTROL   OF   INFECTIOUS   DISEASES  755 

mucous  membrane  as  when  a  well  person  kisses  a  diphtheritic  child; 
conveyance  of  particles  through  the  air,  sprayed  from  the  mouth,  may 
occur,  as  when  a  diphtheritic  patient  coughs  into  an  attendant's  face; 
or  mucous  membranes  may  be  applied  to  skin  or  vice  versa,  as  in  the 
kissing  of  a  smallpox  patient;  but  in  general  the  discharges  are  con- 
veyed somewhat  indirectly.  The  prime  route  from  mucous  membrane 
to  mucous  membrane  is  furnished  by  the  hands.  An  attendant  touches 
the  patient's  lip  or  wipes  out  the  mouth  or  otherwise  performs  toilet 
services  and  receives  the  discharges  upon  his  fingers.  The  fingers  go 
then  to  the  attendant's  mouth  directly,  or  touch  something  (the  tines 
of  a  fork  or  the  bowl  of  a  spoon,  etc.)  which  in  turn  goes  into  his  mouth; 
or  the  attendant  may  touch  the  fork  or  spoon  or  food  of  others  and  thus 
they  become  infected.  He  may  milk  a  cow  and  so  get  the  discharges 
into  the  milk.  With  the  infection  in  his  own  mouth  he  may  kiss 
others  and  transfer  it  to  them.  It  is  impossible  to  outline  the  infinite 
combinations  that  may  occur,  but  the  principles  are  here  made  obvious. 
When  the  infective  discharges  handled  are  those  of  the  bladder  or 
bowel  (as  in  typhoid  fever,  cholera,  etc.)  the  same  dangers  of  trans- 
mission are  encountered  and  unfortunately  too  often  realized.  The 
wholesale  discharge  of  sewage  into  water  supplies  is  merely  a  gross 
example  of  the  same  principle  of  transfer  of  discharges  from  human 
bodies  to  the  human  mouth. 

Another  factor  in  the  transmission  of  disease  (as  distinguished  from 
the  transmission  of  the  germ)  is  the  condition  of  the  inf  ectee.  The  germ 
is  analogous  to  a  seed;  the  methods  of  transmission  are  somewhat 
analogous  to  the  distribution  of  seeds  in  nature;  the  condition  of  the 
infectee  is  analogous  to  the  character  and  nutritional  condition  of  the 
soil  which  the  seed  reaches. 

If  for  any  reason  the  germ  will  not  develop  in  the  soil  where  it  is 
planted,  or,  still  further,  if  it  grows  but  fails  to  produce  those  poisons 
through  which  alone  it  acts,  or  finally  if,  growing  and  producing  its 
poisons,  the  soil  neutralizes  the  poisons,  no  disease  results.  Science, 
logic,  and  the  law  (each  of  which  regards  itself,  and  rightly  so,  as  merely 
an  apotheosis  in  its  own  line  of  " common  sense")  unite  in  the  dictum 
that  a  disease  exists  only  when  the  normal  functions  of  the  body  are  in 
some  way  interfered  with  to  the  detriment  of  the  body.  The  mere  in- 
fection of  the  body  with  a  disease  germ  does  not,  in  science,  logic,  or 
the  law,  constitute  disease.  Hence,  the  reception  of  a  disease  germ 


756   MICROBIOLOGY  OF  DISEASES   OF  'MAN  AND  DOMESTIC  ANIMALS 

into  the  body  is  but  the  first  of  three  essentials,  the  other  two  being 
poison-production  by  the  germ  and  poison-action  on  the  tissues. 
Many  persons  are  insusceptible  to  the  poisons  of  one  or  more  disease 
germs.  In  whatever  way  this  insusceptibility  originate,  (natural, 
acquired  by  a  previous  attack,  or  acquired  by  artificial  treatment) 
the  existence  of  insusceptibilty  tends  to  prevent  the  acquiring  of  the 
disease. 

PRACTICE 

Undoubtedly,  the  one  wholly  efficient  method  of  preventing  the 
spread  of  infectious  diseases  would  consist  in  immunizing  all  the 
possible  infectees  against  all  the  possible  diseases.  Unfortunately, 
we  know  of  no  practical  immunizing  methods  except  in  the  case  of  a 
very  few  diseases,  notably  smallpox  and  typhoid  fever,  paratyphoid 
and  cholera. 

Our  methods  of  control  of  any  disease  therefore  begin  with  the 
attempt  to  destroy  them  at  their  origin  in  the  body  of  the  patient,  but 
such  methods  are  merely  incidental  to  the  destruction  of  the  germs  for 
the  good  of  the  patient  himself,  i.e.,  they  belong  rather  to  therapeusis 
than  to  public  health.  Unfortunately,  also,  scarcely  any  efficient 
method  of  destroying  bacteria  within  the  body  of  the  patient  without 
destroying  the  patient  also  is  known  and  therapeusis  along  this  line 
contents  itself  largely  as  yet  in  so  controlling  the  patient's  condition  as 
to  permit  and  encourage  to  the  highest  the  natural  forces  of  the  body 
to  attack  the  germs.  These  natural  forces,  however,  direct  their 
chief  energies  and  secure  their  chief  results,  not  in  destroying  the  germ 
but  in  neutralizing  the  poisons  the  germs  throw  off,  and  in  practice, 
patients  recover  rather  because  they  have  neutralized  the  poisons  than 
because  they  have  killed  or  ejected  the  germs.  For  this  reason  a 
recovered  patient  often  remains  a  breeding  ground  for  the  germs 
which  caused  the  attack,  but  to  whose  poisons  he  is  now  resistant  or 
immune. 

Practically,  then,  the  germs  must  leave  the  patient's  body  before 
they  can  be  destroyed.  It  is  at  this  stage  that  the  most  efficient  control 
can  be  exercised,  and  that  control  consists  in  killing  them  before  they 
become  scattered.  In  practice  the  efficient  disinfection  of  all  the 
discharges  of  a  patient  will  prevent  the  spread  of  any  disease  from  him. 
But  this  is  not  as  easy  to  do  as  at  first  might  appear.  Ridding  the 


CONTROL   OF   INFECTIOUS   DISEASES  757 

body  of  its  discharges  in  health  is  a  process  dependent  on  the 
individual,  carried  out  by  him  consciously  or  unconsciously  all  his 
life  by  methods  chiefly  directed  to  conserve  convenience  rather  than 
to  prevent  their  spread.  In  health,  the  careless  scattering  of  these 
discharges  is  not  of  great  moment,  but  of  course  the  habits  of  indifferent 
and  careless  discharge,  acquired  in  health,  persist  after  disease  is  con- 
tracted. The  presence  of  an  infective  agent  in  the  discharges  renders 
the  previously  harmless  scattering  of  the  discharges  the  greatest 
menace  that  is  known  to  the  health  of  the  associates.  Hence  one 
primary  requisite  in  the  personal  warfare  against  the  infectious  diseases 
is  to  establish  among  all  people  such  habits  during  health  that  even 
the  normal  discharges  are  not  exchanged.  This  must  be  achieved 
by  teaching  the  individual  not  to  scatter  his  discharges  and  by  teaching 
his  associates  not  to  receive  them,  if  he  does. 

Accepting  conditions  as  they  are,  the  care  of  the  sick  by  watchful, 
well-trained  nurses  who  will  prevent  the  spread  of  the  discharges  must 
largely  take  the  place  of  the  earlier  training  of  the  patient.  Usually 
this  also  is  impossible.  It  would  seem  that  at  least  95  per  cent  of  the 
total  cases  of  infectious  disease  in  this  country  are  cared  for  at  home 
by  the  home  folks,  i.e.,  untrained,  worried,  exhausted  mothers  chiefly, 
trying  to  learn  in  the  actual  face  of  the  enemy,  the  technic  and  knowl- 
edge acquired  quietly  and  systematically  by  the  trained  nurse.  Hence, 
within  the  home,  and  at  present,  sanitary  nursing  to  prevent  spread  of 
disease  is  a  poor  and  often  broken  defence. 

The  third  method  of  control  is  the  destruction  of  the  germs  in  pas- 
sage from  patient  to  prospective  patient;  and  this  must  be  largely  con- 
fined to  the  actual  discharges  when  accumulated  in  one  place;  the  finer 
discharges  thrown  into  the  air  can  hardly  be  followed. 

Under  this  head  may  be  classed  the  disinfection  of  faeces  and  urine, 
the  disinfection  of  bed  clothing,  eating  utensils,  etc.,  coming  into  con- 
tact with  the  patient,  and  especially  the  disinfection  of  the  hands  of 
attendants.  The  throats  of  attendants  often  contain  the  germ,  espe- 
cially when  diphtheria,  scarlet  fever,  measles,  etc.,  are  concerned. 
Unfortunately,  the  disinfection  of  the  throat  is  extremely  difficult  and 
the  scientific  nurse  will  take  every  precaution  to  avoid  receiving  the 
germ  into  the  mouth,  rather  than  try  to  dislodge  or  destroy  it  after 
its  reception.  A  respirator  is  useful  for  this  purpose. 

As  outlined  in  the  preceding  section,  the  principles  involved  in  con- 


75^   MICROBIOLOGY   OF   DISEASES   OF  "MAN   AND   DOMESTIC  ANIMALS 

trolling  infectious  diseases  are  very  simple,  but  in  practice  the  individual 
cannot  be  trusted  to  avoid  spreading  his  discharges,  partly  from  ignor- 
ance, partly  from  carelessness,  often  from  mere  ingrained  bad  habits 
regarding  the  disposal  of  discharges,  especially  those  of  nose  and  mouth, 
indulged  unconsciously  by  those  who  both  know  how  and  mean  to  be 
careful. 

This  would  matter  little  were  the  infected  persons  always  so  sick  as 
to  be  confined  to  the  house  or  to  bed,  especially  if  during  such  confine- 
ment their  discharges  were  under  strict  surveillance  by  scientific  trained 
nurses. 

But  since  many,  perhaps  half,  of  the  infected  persons  are  not  sick 
enough  (if  sick  at  all)  even  to  remain  at  home;  since,  also,  even  severe 
cases,  under  surveillance  in  bed  during  the  height  of  the  attack,  have  a 
prodromal  stage  and  a  convalescent  stage  during  which  they  are  going 
about  although  infective,  it  is  not  hard  to  see  that  the  population  of 
any  community  is  likely  to  embrace  at  any  time  infective  persons  at 
large — persons  who  may  or  may  not  be  aware  of  their  own  condition. 

Theoretically  and  practically,  then,  the  official  control  of  infectious 
diseases  must  begin  with  the  blanket  assumption  that  the  discharges 
of  every  individual  must  be  confined  to  himself  and  especially  prevented 
from  reaching,  through  any  public  utility,  the  mouths  of  other  citizens. 
Official  control  of  the  exchange  by  individuals  of  discharges  within 
the  family  and  in  the  absence  of  any  specific  proof  that  the  discharges 
are  infective,  is  impossible,  although  through  various  agencies  the  indi- 
vidual may  be  urged  to  that  end.  The  moment,  however,  that  the 
individual  or  the  family  engage  in  any  occupation  which  permits  them 
to  inflict  their  discharges  upon  others,  especially  through  food  or  milk, 
that  moment  should  the  individual  or  family  come  under  official 
cognizance,  their  methods  be  inspected  and  their  infectiveness  esti- 
mated. The  same  arguments  apply  to  aggregations  of  individuals 
from  different  families.  So  long  as  private  meetings  are  held,  it  is 
difficult  to  supervise  or  prevent  exchange  of  discharges.  But  public 
and  especially  compulsory  meetings,  at  school,  at  church,  at  theatre, 
etc.,  should  receive  official  attention.  Provision  should  be  made  con- 
cerning all  such  meetings  that  they  be  held  only  in  suitable  places, 
without  overcrowding.  The  exclusion  by  the  officers,  attendants,  or  the 
general  public  of  all  known  to  be  infected  or  suspected  of  infection 
and  of  all  who  more  openly  disregard  ordinary  rules  of  decency  in  the 


CONTROL   OF   INFECTIOUS   DISEASES  759 

disposal  of  discharges  (spitting,  etc.),  should  be  part  of  the  duties  of 
the  health  department. 

Finally,  the  strictest  supervision  of  those  concerned  publicly  and 
officially  in  the  handling  of  public  utilities  on  a  large  scale  (water  sup- 
plies, milk  supplies,  hotels,  restaurants,  food  stores,  etc.)  should  hold  all 
strictly  accountable  for  the  contamination  of  such  supplies  with  dis- 
charges whether  these  be  normal  or  not.  Hence  official  control  of 
infectious  disease  divides  itself  naturally  as  follows: 

1.  The  recognition  and  isolation  of  frank  cases  of  the  diseases  in 
question,  at  home  or,  better,  in  a  proper  hospital. 

2.  The  supervision  of  the  attendants  and  immediate  associates  of 
such  frank  cases. 

(a)  To  detect  among  them  that  one  from  whom  the  frank  case, 
already  recognized,  received  his  infection. 

(b)  To  detect  at  the  earliest  moment  any  other  frank  case  about  to 
develop  from  among  those  associates  who  may  have  been  infected  at  the 
same  time  and  from  the  same  source  as  the  frank  case  already  found. 

(c)  To  prevent  further  spread  from  any  already  infected  associates 
or  those  who  may  become  infected  by  later  association  with  the  frank 
case  during  its  existence  as  such. 

(d)  To  discover  mild,  unrecognized,  and  concealed  cases,  and  carriers. 

3.  The  exclusion  of  all  infected  persons,  their  infected  non-immune 
attendants,  and  immediate  associates,  from  participation  in  public 
life  so  long  as  danger  continues  and  especially  their  exclusion  from  hav- 
ing to  do  with  public  utilities  or  public  gatherings.     Hence  has  arisen 
the   crude   drastic   but   efficient    (when   consistently   and   uniformly 
carried  out  in  every  case)  system  of  isolation  of  the  sick  and  quarantine 
of  his  associates. 

Unfortunately  quarantine  has  become  a  mere  letter-of-the-law  pro- 
cedure, working  great  hardships  on  those  who  conscientiously  sub- 
mit to  it  and  yet  failing  to  achieve  its  objects  because  of  the  great 
number  of  those  who  evade  or  escape  it;  moreover,  because  its  provi- 
sions are  unintelligently  enforced.  Of  what  avail  is  rigid  quarantine 
of  an  infected  family  where  milk  continues  to  be  sold  from  the  same 
premises?  Why  quarantine  the  honest  man  who  has  an  honest 
physician  and  whose  case  is  reported,  while  his  neighbor,  having  the 
same  disease  in  his  family,  calls  no  physician,  or  a  dishonest  one,  and 
therefore  escapes  official  cognizance? 


760   MICROBIOLOGY  OF  DISEASES   OP  MAN  AND  DOMESTIC  ANIMALS 

The  only  remedy  seems  to  be  the  recognition  of  the  principle  that 
harboring  or  having  in  possession  a  case  of  infectious  disease,  unknown 
to  the  proper  officials,  is  a  crime  against  society,  and  that  the  excuse 
that  the  person  harboring  such  case  did  not  know  it  to  be  such  should 
be  of  no  more  weight  than  the  plea  of  ignorance  of  the  law  which  is 
not  allowed  in  other  and  often  far  less  serious  matters. 

The  official  isolation  of  infectious  cases  involves  also  official  responsi- 
bilities regarding  the  release  from  isolation  after  the  acute  attack  is 
over.  Officially  to  declare  a  person  dangerous  to  the  community  does 
no  harm  to  the  community  if  a  mistake  is  made.  An  official  declaration 
that  a  person  is  no  longer  dangerous  and  is  therefore  free  to  enter  into 
the  community  life  again  may,  if  mistaken,  result  in  a  widespread 
outbreak.  No  more  delicate  task  confronts  the  public  health  official 
than  the  making  of  this  decision. 

In  diphtheria,  the  examination  of  cultures  from  the  throat  and  nose 
of  the  person  in  question  and  the  repeated  failure  to  find  the  bacterium 
of  diphtheria  is  usually  considered  a  safe  criterion.  In  scarlet  fever, 
complete  and  continued  restoration  of  the  throat  and  nose  to  normal 
conditions,  together  with  absence  of  ear  discharges,  should  be  required, 
yet  is  not  perfect;  for  it  is  not  very  unlikely  that  the  scarlet  fever 
infective  agent,  whatever  it  may  be,  can  continue  in  a  recovered  scarlet 
fever  throat  as  the  diphtheria  bacterium  may  remain  in  a  recovered 
diphtheria  throat.  In  other  diseases  the  decision  is  based  on  similar 
lines — the  disappearance  of  crusts  in  smallpox  and  chickenpox,  of 
discharges  in  measles,  on  restoration  to  normal  of  whooping  cough; 
but  in  all  these  diseases  the  analogy  with  diphtheria  may  hold  to  a 
greater  or  less  extent.  In  tuberculosis,  the  patient  is  infective  as  long 
as  Bact.  tuberculosis  can  be  found  in  the  sputum;  in  typhoid  fever  the 
patient  is  likewise  infective  as  long  as  the  urine  or  faeces  show  the 
typhoid  bacillus.  In  these  two  diseases,  however,  quarantine  or 
even  isolation  is  not  officially  carried  out  nor  release  from  restriction 
officially  given  to  any  great  extent  or  with  any  marked  uniformity. 

Full  sanitary  nursing  precautions  regarding  a  typhoid  fever  patient's 
discharges  should  continue  for  an  average  of  three  months  after 
recovery. 

PUBLIC  HEALTH  METHODS 

By  request  of  the  editor,  there  are  here  inserted  the  rules  followed^n  the  isola- 
tion and  quarantine  of  the  ordinary  infectious  diseases  in  London,  Canada,  where 


CONTROL    OF    INFECTIOUS   DISEASES  761 

the  writer  was  Medical  Officer  of  Health,  1915-18;  Captain  and  Sanitary  Officer 
to  the  ist  Military  District,  Canadian  Expeditionary  Force,  1916-18;  and  Director 
of  the  Institute  of  Public  Health  since  1912. 

PUBLIC  HEALTH  METHODS,  LONDON,  CANADA,  AS  REVISED  AND  PRO- 
MULGATED BY  THE  INSTITUTE  OF  PUBLIC  HEALTH  OF  WESTERN 

UNIVERSITY,  LONDON,  CANADA 

Public  Health  regulations  are  made  for  the  good  of  the  Public. 
They  are  as  lenient  as  possible,  consistent  with  the  prevention  of  dis- 
ease. They  are  not  made  to  interfere  with  the  individual's  freedom, 
except  as  such  freedom  is  dangerous  to  others. 

I.  HOUSEHOLDER'S  RESPONSIBILITY  TO  THE  BOARD  OF  HEALTH 

1.  Infectious  Diseases: 

It  is  required  that  whenever  any  householder  knows  or  has  reason 
to  suspect  that  any  member  of  the  household  has  any  communicable 
disease,  he  shall  within  twelve  hours  notify  the  Health  Department. 

NOTE. — The  Medical  Officer  of  Health  has  the  right  to  enter  any 
house,  etc.,  in  which  he  knows  or  has  reason  to  suspect  the  presence  of 
any  communicable  disease. 

2.  Births: 

It  is  required  that  every  birth  (including  stillbirths)  shall  be  regis- 
tered by  the  parent  or  guardian,  in  the  prescribed  form,  within  thirty 
days  after  the  date  of  birth. 

3.  Deaths: 

It  is  required  that  every  death  (including  stillbirths)  shall  be 
registered  by  a  member  of  the  household  in  which  the  death  occurs,  in 
the  prescribed  form,  before  a  burial  permit  is  issued. 

II.  PHYSICIAN'S  RESPONSIBILITY  TO  THE  BOARD  OF  HEALTH 

1.  Infectious  Diseases: 

It  is  required  that  whenever  any  physician  knows  or  has  reason  to 
suspect  that  anyone  whom  he  is  called  upon  to  visit  is  infected  with  any 
communicable  disease,  he  shall  within  twelve  hours  notify  the  Health 
Department. 

2.  Births: 

It  is  required  that  every  birth  (including  stillbirths)  be  registered 
by  the  physician  in  attendance,  in  the  prescribed  form,  within  thirty 
days  after  the  date  of  the  birth. 


762    MICROBIOLOGY   OF  DISEASES   OF  MAN   AND   DOMESTIC  ANIMALS 

NOTE. — Further  it  should  be  the  duty  of  every  physician  to  see  that 
every  birth  (including  still-births)  at  which  he  is  in  attendance,  is  prop- 
erly registered  by  the  parent  or  guardian. 

3.  Deaths: 

It  is  required  by  law  that  every  death  (including  stillbirths)  be 
registered,  by  the  physician  last  in  attendance,  on  the  prescribed  form, 
before  a  burial  permit  is  issued. 

NOTE. — Further  it  should  be  the  duty  of  the  physician  to  state  the 
cause  of  death  clearly  and  in  accordance  with  the  ''International  List 
of  Causes  of  Death." 

III.  PENALTIES 

1 .  Any  person  (householder  or  physician)  whose  responsibility  it  is, 
by  the  Statutes  of  Ontario,  to  report  a  case  of  any  communicable  dis- 
ease, and  who  neglects  to  do  so,  may  incur  a  penalty  not  less  than  $25.00 
nor  exceeding  $100.00. 

2.  Any  person  required  by  the  Statutes  of  Ontario  to  register  a 
birth  or  death,  and  who  neglects  to  do  so,  may  incur  a  penalty  not 
exceeding  $10.00. 

3.  Any  person  who  wilfully  makes  or  causes  to  be  made  a  false 
statement  touching  any  of  the  particulars  required  in  registering  a 
birth  or  death  may  incur  a  penalty  of  $50.00. 

3.  Any  person  who  wilfully  makes  or  causes  to  be  made  a  false 
statement  touching 'any  of  the  particulars  required  in  registering  a 
birth  or  death  may  incur  a  penalty  of  $50.00. 

IV.  LIST  OF  COMMUNICABLE  DISEASES  WHICH  MUST  BE 
REPORTED  IN  ONTARIO 

Smallpox  Anterio  Poliomyelitis  German  Measles 

Leprosy  (Infantile  Paralysis)  Glanders 

Scarlet  Fever  Cerebro-spinal  Meningitis  Anthrax 

Diphtheria  Typhoid  Fever  Tuberculosis  (of  all  forms) 

Bubonic  Plague  Chickenpox  Rabies  (Hydrophobia) 

Asiatic  Cholera  Whooping  Cough  Erysipelas 

Measles  Mumps  Any  other  communicable 

Syphilis  Gonorrhea  disease 

V.  PERMITS  TO  LEAVE  QUARANTINE,  ETC. 

i.  The  immune  contacts  and  others,  who  under  the  schedule,  may 
be  allowed  to  attend  business,  school,  etc.,  despite  quarantine  on  other 
members  of  the  household,  MUST  HAVE  WRITTEN  PERMITS  from 


CONTROL   OF   INFECTIOUS   DISEASES  763 

the  M.  O.  H.  so  to  do.     Recovered  patients  cannot  return  to  school 
or  business  or  otherwise  escape  quarantine  without  such  permits. 

2.  The  attending  physician,  while  legally  bound  to  report  all  in- 
fectious diseases,  cannot  legally  impose  or  release  quarantine.  No 
one  but  the  M.  O.  H.  can  legally  impose  or  release  quarantine. 

VI.   METHODS  OF  HANDLING  THE  INFECTIOUS   DISEASES — LONDON, 
CANADA,  JANUARY,  1916 

1.  Tuberculosis: 

All  cases  in  all  stages  are  reportable  within  twelve  hours  of  dis- 
covery, to  the  M.  O.  H.  who  is  thereafter  legally  responsible  for  the 
prevention  of  spread  of  the  disease. 

In  all  non-infectious  stages,  this  supervision  is  restricted  to  watch- 
ing— through  the  attending  physician,  if  there  be  one — the  progress  of 
the  case  in  order  to  detect  the  development  (if  any)  of  an  infectious 
stage. 

There  are  no  restrictions  other  than  the  above  on  the  patient  dur- 
ing non-infectious  stages. 

In  all  infectious  stages,  definite  arrangements  must  be  made  to 
prevent  infection:  these  may  be  made  through  the  attending  physician, 
if  there  be  one;  otherwise  directly  with  the  relatives  or  patient. 

There  are  no  restrictions  on  the  associates  of  the  patient.  There- 
fore placarding  and  other  quarantine  measures  are  not  necessarily 
imposed.  Terminal  disinfection,  by  Board  of  Health,  is  free  in  tuber- 
culosis. It  is  not  performed  in  other  diseases,  except  under  special 
circumstances. 

2.  Epidemic  Anterior  Poliomyelitis  (Infantile  Paralysis): 

All  cases  are  reportable  in  the  acute  stage.  The  patient  must  be 
isolated  during  the  continuance  of  fever:  and  the  usual  precautions 
relating  to  disinfection  of  discharges  must  be  carried  out  during  the 
progress  of  the  disease. 

3.  Epidemic  Cerebro-spinal  Meningitis: 

As  in  poliomyelitis,  with  the  addition  that  nurses,  etc.,  shall  wear 
respirators  during  the  performance  of  intimate  personal  services  to  the 
patient. 

4.  Rabies: 

Persons  bitten  by  dogs  suspected  of  Rabies  should  receive  the 
Pasteur  Treatment,  furnished  by  the  M.  O.  H.  through  the  Provincial 


764   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

Board  of  Health.  Dogs  who  have  bitten  persons  should  not  be  killed 
but  should  be  securely  confined  for  observation.  If  surviving  a  week 
the  diagnosis  of  not  Rabies  is  established. 

5.  Venereal  Diseases: 

Under  Provincial  legislation  passed  in  1918,  everyone  (except  lay 
householders)  who  is  responsible  under  other  laws  to  report  other 
infectious  diseases  is  required  to  report  venereal  diseases,  but  the 
patient's  name  is  not  to  be  given  (serial  numbers  to  be  used  instead) 
unless  the  patient  abandons  treatment,  whereupon  full  identification 
data  must  be  sent  the  Medical  Officer  of  Health,  who  must  then  fol- 
low up  the  case  and  take  proper  methods  for  prevention  of  spread. 
The  legislation  is  very  detailed  and  complete.  Military  and  civil 
cooperation  is  assured  also. 

6.  The  other  Rarer,  Infectious  Diseases,  which  are  reportable: 
Anthrax,  Asiatic   Cholera,   Bubonic   Plague,    Glanders,  Leprosy, 

Erysipelas,  etc.,  will  be  handled  as  they  arise  according  to  the  circum- 
stances of  the  case. 

7.  The  More  Common  Infectious  Diseases: 

Smallpox,  Scarlet  Fever,  Measles,  Diphtheria,  Typhoid  Fever, 
Chickenpox,  Whooping  Cough,  Mumps  and  German  Measles  are 
handled  according  to  a  schedule  given  beyond. 

Free  Diphtheria  antitoxin,  etc.,  is  supplied  on  application  to  the 
Medical  Officer  of  Health. 

DEFINITIONS 

" Cases"  are  persons,  sick  of  the  disease  in  question.  Those  ex- 
posed to  an  infected  person,  during  an  infectious  stage,  whether  that 
infected  person  be  sick  (a  "case")  or  well  (a  " carrier"),  are  known  as 
" contacts."  The  contacts  may  be  immune  or  not,  infected  or  not. 
It  is  the  fact  of  exposure  to  infection  that  makes  them  " contacts." 

The  term  "isolation"  means  restriction  of  freedom  of  an  internally 
infectious  person,  i.e.,  of  a  person  sick  with  the  disease  (a  "case"),  or 
of  a  person,  whether  immune  or  not,  who  is  well  but  infectious,  and 
therefore  is  a  "carrier."  A  non-immune  "carrier"  may  become  a 
"case,"  if  the  disease  develops  in  him  later. 

The  term  "quarantine"  is  properly  applied  only  to  restriction  of 
freedom  of  non-immune  well  persons  who  have  been  so  exposed  to  in- 
fected persons  as  to  make  it  likely  that  they  themselves  may  develop 


CONTROL   OF   INFECTIOUS   DISEASES  765 

the  disease.  Neither  isolation  nor  quarantine  can  be  justified  if  im- 
posed on  non-infectious  immunes,  or  on  non-immunes  who  have  not 
been  exposed. 

RULES  FOR  RELEASE  OF  "  CASES  "  FROM  ISOLATION 

The  case,  whether  isolated  at  a  hospital,  at  home  with  a  trained 
nurse  in  attendance,  or  at  home  without  a  nurse,  will  be  set  free,  and 
may  go  to  school,  work,  etc.,  only  after  full  recovery  as  determined 
by  the  M.  O.  H.,  and  in  addition  as  follows: 

In  Smallpox,  after  all  scales,  plaques,  crusts,  etc.,  have  disappeared, 
as  determined  by  the  M.  O.  H. 

In  Scarlet  Fever,  not  less  than  six  weeks  from  onset  and  then  only 
if  temperature,  nose,  throat,  ears,  etc.,  have  been  normal  for  one  week, 
no  discharge  of  any  kind  from  any  orifice  exists,  and  all  wounds,  sores, 
herpes,  etc.,  are  completely  healed. 

In  Measles  proper,  on  the  fourteenth  day  from  onset,  which  will  be 
the  tenth  day  from  beginning  of  rash. 

In  Diphtheria,  after  three  consecutive  negative  cultures  have  been 
obtained  from  both  nose  and  throat. 

In  Typhoid  Fever,  eight  weeks  after  onset. 

In  Chickenpox,  after  all  crusts,  scales,  scabs,  etc.,  have  disappeared. 

In  Whooping  Cough,  one  week  after  last  whoop,  or  six  weeks  from 
first  whoop,  whichever  comes  first. 

In  Mumps,  three  weeks  from  onset. 

In  German  Measles,  one  week  from  onset. 

PLACARDING  OF  HOUSE — EXTERNAL 

If  the  case  goes  to  a  hospital,  no  placard  is  placed  on  the  home  from 
which  he  came,  notwithstanding  that  exposed  non-immunes  may  be 
quarantined  there,  unless  the  quarantine  is  not  properly  observed; 
whereupon  a  placard  may  be  used  in  any  disease,  or  a  special  police- 
man detailed  to  watch  the  house  or  both. 

If  the  case  remains  at  home,  where  the  family  occupies  a  house, 
both  front  and  rear  entrances  are  placarded  in  certain  diseases  (Small- 
pox, Scarlet  Fever,  Measles,  Diphtheria  and  the  "  Rarer  Infectious 
Diseases"  mentioned  above,  under  the  Provincial  Law;  in  certain 
others,  typhoid  fever,  chickenpox,  whooping  cough,  mumps,  German 
measles,  placarding  is  optional  and  is  enforced  only  if  isolation  or 


766   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

quarantine  is  evaded  by  the  inmates.  In  hotels  or  apartment  houses, 
only  that  room,  suite  or  apartment  occupied  by  the  patient  and  his 
family  need  be  placarded;  otherwise  the  same  rules  should  be  followed. 
Milk  men  are  warned  that  they  may  continue  delivery,  but  must 
take  no  bottle  or  other  receptacle  away  until  the  placard  is  removed, 
and  that  all  such  receptacles  must  be  sterilized  before  use. 

PLACARDING  OF  HOUSE — INTERNAL 

Under  whatever  circumstances  isolation  or  quarantine  is  carried 
out  in  houses,  hotels,  apartments,  or  elsewhere,  except  in  contagious 
hospitals,  there  is  fixed  to  an  internal  wall  at  a  convenient  point  a 
notice,  giving  the  general  rules  regarding  who  are  isolatable,  who  are 
quarantinable,  who  may  go  free,  and  the  actual  names  of  all  inmates 
are  distributed  in  writing  into  these  three  groups  on  the  notice.  For 
all  who  may  go  free,  permits  in  writing  are  furnished  and  the  fact  that 
permits  to  go  back  and  forth  have  been  issued  is  also  recorded  on  this 
notice. 

We  believe  the  internal  placard,  properly  filled  out  as  above,  to  be 
of  the  greatest  educational  value;  it  brings  everything  down  to  " brass 
tacks,"  inasmuch  as  the  family  is  classified  as  to  immunity  or  non- 
immunity,  freedom  or  restriction,  by  name  in  writing  upon  it,  and 
because  there  can  therefore  be  no  misunderstanding  on  the  part  of  any 
individual  concerned. 

In  the  writer's  opinion,  an  internal  placard  should  be  used  in  all 
infectious  diseases  to  the  exclusion  usually  of  the  external  placard;  the 
latter  being  used  only  when  the  rules  laid  down  in  the  internal  placard 
are  broken  wilfully  by  the  inmates. 

QUARANTINE  PERIODS  FOR  CONTACTS 

Contacts  are  those  persons  who  come  into  such  close  relationships 
with  an  infected  person,  during  an  infectious  stage,  as  may  reasonably 
be  held  to  afford  opportunity  for  the  transfer  of  the  infection  from  the 
infected  person  to  the  other.  The  existence  of  such  opportunity  for 
infection  by  no  means  ensures  its  occurrence,  but  in  most  diseases  there 
is  no  sure  way  of  determining  whether  or  not  infection  has  occurred 
other  than  by  watching  to  see  if  the  disease  develops.  This  period  of 
observation  is  usually  the  period  of  quarantine.  In  diphtheria,  con- 
tacts may  be  examined  by  taking  a  culture  from  nose  and  throat.  If 


CONTROL    OF   INFECTIOUS   DISEASES  767 

the  culture  prove  negative  the  contact,  whether  immune  or  not,  may 
be  set  free  because,  since  he  is  not  infected,  he  will  not  become  sick 
himself  nor  can  he  give  the  disease  to  others.  If  it  prove  positive,  the 
contact,  whether  immune  or  not,  is  dangerous,  even  though  not  sick, 
and  should  be  isolated  as  dangerous;  many  physicians  advocate  giving 
a  prophylactic  dose  of  antitoxin  (1000  units)  to  prevent  the  develop- 
ment of  the  disease  in  such  contact.  This  dose  ensures  the  non-develop- 
ment of  the  disease  for  two  weeks,  at  which  time  it  must  be  renewed  if 
protection  is  still  desired.  Prophylactic  doses,  indeed,  even  therapeu- 
tic doses  (10,000  to  50,000  units),  do  not  affect  the  bacilli  themselves, 
and  therefore  the  infected  contact  who  has  been  protected  against 
the  disease  by  antitoxin  so  far  as  his  own  health  is  concerned,  never- 
theless remains  a  menace  to  others  so  long  as  the  bacilli  remain  in  nose 
or  throat.  The  practice  of  releasing  an  infected  contact  as  soon  as  he 
is  immunized  is  illogical,  unjustifiable,  absurd  and  dangerous.  Simi- 
lar tests  of  nose  and  throat  (by  smears  instead  of  culture)  may  be  made 
in  the  case  of  Cerebro-spinal  Meningitis,  the  handling  of  the  contacts 
and  the  conduct  of  release  or  isolation  being  similarly  carried  out  on 
the  basis  of  the  results.  Immune  contacts  (unless  determined  to  be 
infective  as  above)  may  be  released  at  once,  after  disinfection  of  their 
hands;  and  should  be  warned  against  the  dangers  of  acquiring  and 
carrying  the  infection  on  their  hands  (in  poliomyelitis,  cerebro-spinal 
meningitis,  influenza,  and  diphtheria,  in  their  throats  and  noses  also) 
as  a  result  of  further  contact  with  the  case,  or  with  carriers. 

Non-immune  contacts  should  be  questioned  carefully  as  to  their 
dates  of  contact  with  the  case;  and  the  dates  of  infectiveness  of  the 
patient  should  be  compared  with  these,  in  order  to  determine  when  the 
exposure  began  and  when  it  ceased.  If  minute  enquiry  of  this  kind 
cannot  be  made,  or  is  unsatisfactory  for  any  reason,  it  is  proper  to 
assume,  until  proved  otherwise,  that  members  of  the  same  household, 
office,  etc.,  were  all  exposed  on  the  first  day  the  case  became  infectious, 
ard  continued  to  be  exposed  daily  up  to  the  date  of  isolation  of  the  case. 

Whether  exact  individual  determinations  of  these  dates  of  exposure 
be  made,  or  the  blanket  assumption  indicated  be  applied,  the  further 
calculations  are  as  follows:  To  the  date  of  first  exposure  (usually  the 
date  of  onset  in  the  patient)  add  the  minimum  incubation  period  of 
the  disease  in  question :  this  will  indicate  the  earliest  date  at  which  any 
infected  contact  can  develop  the  disease  and  therefore  the  beginning  of 


768   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

quarantine  (or  observation)  necessary  for  such  contact  or  contacts. 
To  the  date  of  last  exposure  (usually  the  date  of  isolation  of  the  patient) 
add  the  maximum  incubation  period  of  the  disease  plus  the  maximum 
prodromal  period,  thus  fixing  the  last  day  on  which  any  contact  will 
develop  the  typical  symptom  of  disease  and  hence  the  end  of  the  neces- 
sary quarantine  (or  observation)  period.  Theoretically  the  maximum 
incubation  period  would  be  sufficient  but  the  prodromal  period  is  in- 
cluded in  practice  beca'use,  in  many  diseases,  the  onset  may  be  trivial 
and  may  therefore  be  overlooked,  the  contact  being  discharged  just  as 
he  begins  to  be  infective;  if  the  full  prodromal  period  be  included,  the 
most  careless  can  hardly  fail  to  observe  that  the  disease  was  begun 
and  that  isolation  must  be  carried  out. 

In  those  diseases  where  the  incubation  period  is  long,  and  is  not  in- 
fectious (for  example,  in  mumps),  there  will  often  be  found  to  exist  a 
period  after  isolation  of  the  original  patient  and  before  the  first  contact 
can  become  sick,  of  a  week  or  even  ten  days  or  so.  During  this  period 
non-immune  contacts  may  be  given  interim  permits,  allowing  them  to 
go  on  with  their  usual  lives  up  to  the  date  when  the  first  case  may  be 
expected  to  develop.  On  that  date  they  should  be  quarantined  (or 
placed  under  observation)  for  the  necessary  period. 

In  measles,  and  German  measles  similar  methods  may  be  followed. 
In  Diphtheria  and  Scarlet  Fever  it  is  rare  that  the  time  so  saved  to  the 
contact  is  long  enough  to  be  worth  the  extra  risk  and  work  involved. 


OBSERVATION  VERSUS  QUARANTINE 

In  dealing  with  intelligent  conscientious  people,  or  with  people 
under  full  control  as  in  schools,  armies,  etc.,  non-immune  contacts,  if 
observed  twice  daily  by  competent  medical  men  or  nurses,  throughout 
the  period  during  which  they  may  develop  the  disease,  may  be  freed  of 
all  other  quarantine  restrictions.  The  family  physician  will  generally 
cooperate  with  the  Health  Officer  in  this  twice-daily  inspection,  thus 
saving  much  otherwise  necessary  restriction,  to  the  great  satisfaction 
of  all  concerned. 

The  quarantine  periods  and  examples  of  calculations  are  appended. 
See  Table. 


CONTROL   OF   INFECTIOUS   DISEASES 


769 


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(severe) 

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.a 

Diphtheria  

Typhoid  

Para-typhoid... 

Cerebro-spinal. 

Poliomyelitis.  . 

*  Average  14 

4'J 


770   MICROBIOLOGY   OF  DISEASES   OF  MAN  AND   DOMESTIC  ANIMALS 

REGULATIONS  REGARDING  VISITORS 

Persons  of  all  ages  and  both  sexes,  who  are  not  immune  to  the 
disease  in  question,  must  be  rigidly  excluded  from  all  houses  where  cases 
or  non-immune  contacts  are  isolated  or  quarantined. 

Immunes  are  rigidly  excluded  from  contact  with  the  patient — in- 
deed no  one  should  see  the  patient  except  physician,  nurse  or  other 
attendant,  although  the  law  admits  the  clergy,  and  of  course  under- 
takers, in  case  of  death. 

Where  important  affairs  make  it  essential  that  a  visitor  other  than 
those  permitted  by  law,  should  interview  the  case,  or  otherwise  come 
in  contact  with  him,  immunes  should  be  selected  to  make  the  visit, 
and-  they  should  be  required  to  obtain  written  permits  from  the  M. 
O.  H.  and  be  guided  in  their  conduct  during  and  after  the  interview 
by  the  general  rules  such  as  are  followed  by  doctors  and  nurses  to  avoid 
carrying  infection  to  others. 

IN  CASE  OF  DEATH 

The  body  should  be  handled  only  by  a  skilled  undertaker:  the 
orifices  should  be  antiseptically  plugged  and  the  body  wrapped  in  a 
sheet  wrung  out  of  bichloride  solution,  i  in  500.  Metallic  coffins, 
embalming,  etc.,  are  entirely  unnecessary,  merely  adding  to  the  ex- 
pense of  the  stricken  family,  without  affording  any  additional  protection 
to  the  public  health.  If  such  unnecessarily  refined  precautions  are 
insisted  upon  by  the  public  health  authorities,  then  the  community, 
not  the  individual,  should  bear  the  expense. 

The  funeral  should  be  private,  only  members  of  the  household 
already  in  the  house  or  proved  immunes  from  outside  being  permitted 
to  attend  any  ceremonies  in  the  house. 

After  the  house  ceremonies  are  over,  non-infectious  members  of  the 
family,  immune  or  not,  may  attend  the  funeral  procession;  and  whoever 
likes  to  do  so  may  join  the  procession  outside  the  house  and  go  with  it 
wherever  they  please. 

Local  laws  and  ordinances  often  call  for  excessive  precautions  and 
of  course  must  be  obeyed.  The  above,  however,  include  all  the  real 
essentials  for  health  protection. 


CONTROL  OF  INFECTIOUS  DISEASES  771 

DISINFECTION 

Two  systems  of  disinfection  have  been  long  recognized,  concurrent 
and  terminal.  The  former  concerns  the  daily,  hourly  attention  to, 
and  disinfection  of,  everything  coming  in  contact  with  the  patient, 
especially  with  his  discharges  and  all  that  they  may  contaminate. 
The  latter  concerns  the  final  disinfection  of  the  patient's  room,  per- 
haps of  the  whole  house,  occupied  by  him  during  the  attack,  after  the 
recovery  of  the  patient. 

Very  much  undue  emphasis  has  been  given  to  terminal  disinfection. 
Large  expenditures  are  made  for  this  purpose  and  great  faith  placed  in 
it,  unfortunately  to  the  exclusion  of  attention  to,  and  reliance  on,  the 
infinitely  more  useful  and  logical  concurrent  disinfection,  which, 
properly  done,  ought  almost  wholly  to  displace  it. 

Terminal  disinfection  should  be  done  following  tuberculosis  of  the 
lungs,  anthrax  and  plague;  in  tuberculosis  because  of  the  great  numbers 
and  wide  distribution  of  the  bacteria  thrown  out  by  the  patient, 
especially  the  careless  patient;  in  anthrax  because  of  the  existence  of 
resistant  spores  possibly  attached  to  furniture,  etc. ;  in  plague  because 
of  the  intense  virulence  of  the  organism  and  its  tendency,  like  anthrax, 
to  infect  directly  through  the  skin.  In  the  ordinary  diseases  of  the 
temperate  zone,  however,  terminal  disinfection  cannot  for  a  moment 
take  the  place  of  concurrent  disinfection  and  is  unnecessary  if  the 
former  be  properly  carried  out. 

METHODS  OF  DISINFECTION 

CONCURRENT  DISINFECTION. — The  disinfection  of  infected  discharges,  and  of 
everything  coming  into  contact  with  the  discharges,  whether  the  discharges  be  of 
the  nose,  mouth,  bladder,  or  bowel,  and  whether  the  things  which  come  into  con- 
tact with  the  discharges  be  utensils,  clothing,  hands,  furniture,  etc.,  should  be  done 
at  once,  as  soon  as  the  discharges  appear,  or  the  articles,  hands,  etc.,  become 
contaminated. 

Bladder  and  bowel  discharges  deposited  directly  in  proper  sewer-connected 
toilet-bowls  require  no  disinfectant  treatment;  but  the  seat,  door-knobs,  toilet 
paper  rack,  flush  pull  and  so  on,  which  the  discharges  may  reach,  directly  or  through 
the  patient's  hands,  should  receive  disinfection  every  time  the  toilet  is  used  by  such  a 
patient.  Where  bed-pans  or  urinals  are  used  and  then  emptied  into  such  a  toilet- 
bowl,  disinfection  should  be  done  of  the  hands  of  the  attendant  who  empties  the 
pan,  of  the  whole  pan  itself,  and  of  any  part  of  seat  or  bowl  (not  reached  by  the 
flush)  contaminated  by  splash  or  dribbles  from  the  bed-pan  or  urinal. 


772    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

Where  outdoor  toilets  or  indoor  toilets  not  connected  with  a  sewer  are  in  use  the 
discharges  must  always  be  disinfected — preferably  by  half-filling  the  bed-pan  or 
urinal,  before  use,  with  a  saturated  solution  of  milk  of  lime  (unslaked  lime,  in  water, 
to  saturation — cool  and  pour  off  the  liquid  parts)  into  which  the  discharges  are 
received.  Where  such  toilets  are  used  by  the  patient  directly,  an  abundant  layer 
of  powdered  unslaked  lime  should  cover  the  discharges  as  soon  as  they  are  de- 
posited. Such  layer  should  be  an  inch  deep.  Precautions  regarding  the  seats,  door- 
knobs, hands,  etc.,  should  be  followed  as  above  described.  The  difficulty  in  en- 
forcing these  precautions  makes  fly-screening  a  better  plan. 

Soiled  bed  clothing  or  other  clothing,  handkerchiefs,  etc.,  may  be  rolled  up  and 
placed  directly  in  boiling  water;  but  if  some  interval  must  elapse  before  they  can  be 
boiled,  they  should  be  put  directly  into  5  per  cent  carbolic  acid  solution,  or  o.i  of  i 
per  cent  bichloride  of  mercury  solution  or  other  disinfectant  of  similar  killing  power 
for  at  least  half  an  hour.  Thereafter  they  may  be  handled  as  uninfected  clothing. 

Eating  utensils  after  use  should  go  directly  into  boiling  water  for  several  minutes 
and  then  be  washed  in  the  ordinary  way.  Spoons  used  for  medicine,  toys,  ther- 
mometers, etc.,  which  it  may  be  inconvenient  or  impossible  to  put  into  boiling  water, 
should  be  immersed  in  5  per  cent  carbolic  acid  or  o.i  per  cent  bichloride  solution  for 
half  an  hour,  then  washed. 

These  solutions  may  be  used  also  for  the  hands  and  a  large  bowl  of  one  or  both  of 
them  (carefully  labelled,  and  out  of  reach  of  children,  etc.)  should  be  constantly 
ready;  into  this  the  patient's  and  attendant's  hands  should  be  dipped  after  every 
contamination. 

Discharges  from  the  nose  and  mouth  should  be  collected  on  paper  or  rags  and 
burned  at  once.  If  inconvenient  to  burn  them,  they  should  be  dropped  into  carbolic 
or  bichloride  solutions  as  above,  and  disposed  of  as  harmless  after  a  half-hour's 
soaking. 

It  is  difficult  to  specify  every  form  of  contact  to  be  guarded  against  by  disin- 
fection, but  the  foregoing  are  the  chief  ones  to  watch  for,  and  the  principles  given 
should  be  widely  and  intelligently  applied — remembering  always  that  the  dis- 
charges contain  the  danger. 

TERMINAL  DISINFECTION. — Sulphur  disinfection  (4  pounds  burned  for  every 
1,000  cubic  feet  of  space,  in  the  presence  of  steam  sufficient  to  saturate  the  atmos- 
phere) is  effective  for  disease  bacteria — also  for  roaches,  bedbugs,  etc.,  and  for 
mice,  rats,  etc.  But  it  injures  fabrics  by  bleaching  them,  and  metals  by  tarnishing 
them.  Formaldehyde  vapor  is  now  used  in  its  place  for  disinfection;  but  flies, 
bedbugs,  etc.,  are  not  successfully  exterminated  thus.  The  most  recent  approved 
method  for  use  in  the  disinfection  of  houses  is  the  Minnesota  State  Board  of 
Health  potassium  permanganate  formaldehyde  method. 

For  each  1,000  cubic  feet  of  space  the  following  should  be  used: 

Potassium  permanganate  (crystals) 1 1  ounces 

Solution  formaldehyde  (U.  S.  P.  1900) n  ounces 

Water. . .  9  ounces 


CONTROL   OF   INFECTIOUS   DISEASES  773 

Directions  for  use: 

Prepare  the  room  to  be  disinfected  by  sealing  all  cracks,  windows,  ventilators, 
etc.,  and  all  the  doors  but  the  one  for  exit,  with  wet  newspaper  strips;  open  all 
blankets,  drawers,  etc.;  separate  and  open  up  all  books,  clothing,  etc.,  in  the  room. 
Have  wet  strips  of  paper  in  readiness  to  seal  the  last  door  when  the  disinfection 
has  been  started  and  the  operator  has  left  the  room.  The  windows  should  be  left 
unlatched  so  that  they  may  be  opened  from  the  outside  after  the  disinfection  is 
completed. 

Use  a  metal  pail  with  lapped  (not  soldered)  seams,  or  an  earthenware  receptacle, 
holding  not  less  than  fourteen  (14)  quarts,  in  which  to  mix  the  above  ingredients. 
Place  the  receptacle  on  bricks  standing  in  a  pan  of  water,  but  the  receptacle  should 
not  touch  the  water. 

Place  the  1 1  ounces  of  potassium  permanganate  in  the  receptacle,  distributing  it 
evenly  over  the  bottom. 

Mix  the  formaldehyde  (n  ounces)  and  the  water  (9  ounces),  and  pour  this 
mixture  over  the  potassium  permanganate  in  the  receptacle. 

This  done,  the  operator  should  leave  the  room  as  quickly  as  possible,  sealing  the 
door  behind  him  with  the  wet  strips  of  paper  prepared  in  advance  for  this  purpose. 

The  directions  above  apply  to  the  disinfection  of  a  room  containing  1,000  cubic 
feet  or  less.  If  a  room  contains  more  than  1,000  cubic  feet  of  space,  use  one  of  the 
above  disinfecting  outfits  for  each  1,000  cubic  feet  or  fraction  thereof.  Do  not 
attempt  to  use  a  double  charge  in  a  container  of  even  double  capacity. 

In  disinfecting  a  whole  house,  begin  with  the  most  distant  room  and  having 
mixed  the  potassium  permanganate,  formaldehyde,  and  water  in  the  proper  re- 
ceptacle, close  the  door  of  the  room  and  seal  it  at  once  as  directed  above.  Proceed 
in  this  way  in  the  disinfection  of  all  the  rooms.  Leave  the  seals  unbroken  on  the 
window  and  doors  for  six  hours,  after  which  the  rooms  should  be  opened  up  and 
thoroughly  aired.  The  temperature  of  the  room  at  the  time  of  disinfection  should 
not  be  below  7o°F. 

No  paper,  cotton,  cloth,  wood,  or  other  combustible  material  should  be  in  or  near 
the  disinfecting  outfit  for  fear  of  fire,  and  no  flame  should  be  permitted  in  the  room 
near  the  disinfecting  outfit. 

CARRIAGE  or  INFECTION  BY  BIOLOGICAL  AGENTS 

The  transmission  of  yellow  fever  and  malaria  by  mosquitoes,  in  the 
course  of  which  the  parasite  causing  the  disease  must  undergo  a  whole 
series  of  developmental  changes  before  the  mosquito  can  become  infective, 
is  now  well  understood.  But  the  mechanical  carriage  of  infec- 
tious material  by  flies  from  privy  vaults  or  bed  pans  or  even  mucous 
membranes  or  open  wounds  to  food  and  drink  or  to  other  mucous 
membranes  or  wounds  has  not  been  very  long  established. 

That  typhoid  fever  and  dysentery  have  many  times  occurred  in 
epidemic  form  chiefly  by  the  carriage  of  the  infective  agents  by  flies  the 


774   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

writer  firmly  believes  as  the  result  of  personal  investigation,  as  well  as 
from  the  reports  of  others.  Similar  mechanical  carriage  of  infection  on 
the  outside  of  the  body  has  been  attributed  to  rats,  dogs,  cats,  even  to 
cows  and  horses.  This  must  not  be  confused  with  the  dissemination  of 
certain  diseases  by  horses  actually  sick  with  the  disease  (glanders)  or 
carrying  the  germs  in  their  intestines  (tetanus),  by  cows  actually  sick  of 
tuberculosis,  or  by  other  similar  instances  of  disease  derived  directly 
from  preceding  cases  or  carriers  in  the  lower  animals. 

Another  class  of  cases  where  lower  animals  convey  disease  by  biting, 
and  yet  act  merely  mechanically  is  instanced  by  the  septicaemia  some- 
times arising  from  bites  of  well  animals  (rats,  snakes,  mosquitoes,  etc.), 
the  bite  acting  merely  to  admit  to  the  tissues  pathogenic  forms  acci- 
dentally present  in  the  animal's  mouth  or  on  the  skin  of  the  bitten 
person.  These  must  be  distinguished  from  cases  where  the  animal 
transmits  thus  a  disease  from  which  it  is  itself  suffering  (as  when  a  rabid 
dog  spreads  'rabies  by  biting  other  animals  or  man)  and  from  true 
poisoning  by  injection  of  animal  products  at  the  time  of  biting  (as  done 
by  poisonous  snakes,  mosquitoes,  etc.). 


CHAPTER  VI 
MICROBIAL  DISEASES  OF  MAN  AND  DOMESTIC  ANIMALS 

DISEASES  CAUSED  BY  MOLDS*  AND  YEASTS 

The  diseases  produced  by  fungi  in  higher  animals  are  mostly 
localized  infections  of  the  skin  (dermatomycoses),  of  the  mouth  and 
throat  (thrush),  of  the  lungs  and  air  passages  (pneumomycoses),  and 
of  the  lymphatics  (Sporotrichosis  and  Saccharomycosis). 

PNEUMOMYCOSIS  f 

ASPERGILLOSIS. — The  fungus  disease  of  the  lungs  and  air  cells  of 
birds  is  quite  uniformly  attributed  to  Aspergillus  fumigatus  which  is 
widely  distributed  in  the  soil  and  upon  feed  and  grains.  The  agency  of 
this  species  in  causing  disease  is  well  established.  It  grows  best  at 
blood-heat.  Inoculation  experiments  have  produced  the  disease  in 
animals.  Isolated  cases  are  recorded  in  which  this  organism  is  regarded 
as  the  cause  of  disease  in  horses  or  cattle  and  even  man.  Biologic 
forms  of  A.  fumigatus  are  very  widely  distributed.  Many  of  them  are 
readily  separated  by  cultural  characters.  Pathogenicity  is  not  lim- 
ited to  one  or  a  few  strains  since  lesions  have  been  described  as  due  to  a 
wide  range  of  the  varieties  of  this  group.  Other  species  of  Aspergillus, 
A.  flavus,  A.  nidulans,  A.  niger,  have  been  listed  among  pathogenic 
forms  from  their  presence  at  times  in  diseased  tissue.  Whether  these 
species  are  ever  a  primary  cause  of  disease  is  doubtful,  but  their  pres- 
ence and  activity  in  such  infected  areas  has  been  established. 

Secondary  Infections. — Spores  of  any  species  of  fungus  found  in 
the  locality  may  find  lodgment  in  wounds,  orifices  open  to  the  outside, 
such  as  the  external  ear  or  the  air  passages.  Many  of  these  spores  will 
germinate  in  such  situations.  If  favored  by  dirt,  pus,  mucus,  or  exist- 
ing pathological  condition  the  resulting  growth  in  some  species  de- 
velops into  a  secondary  infection;  most  species  lack  entirely  the  power 
to  produce  disease.  The  appearance  of  molds,  especially  species  of 

*  Arranged  ^enerically  as  far  as  possible, 
t  Prepared  by  Charles  Thorn. 

775 


776   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC   ANIMALS 


Mucor,  Penicillium,  and  Aspergillus,  in  such  situations  has  been  fre- 
quently reported  in  literature.  In  very  large  measure  at  least  such 
presence  may  be  regarded  as  evidence  of  lack  of  care,  cleanliness,  of 
even  ordinary  precautions  when  the  infection  involves  man. 

THRUSH* 

The  parasite  of  thrush,  Oidium  albicans  Robin,  (Saccharomyces  albi- 
cans,  Reiss),  in  culture  produces  a  scanty  mycelium,  submerged  in  the 


FIG.  161. — Oidium  albicans,  from  a  culture  obtained  from  Krai. 

substratum,  which  branches  monopodially.  The  tendency  to  budding 
and  to  the  entire  suppression  of  the  mycelium  leads  some  to  regard  this 
form  as  a  yeast  (Fig.  161).  It  attacks  the  mucous  membrane  of  the 


*        FIG.  162. — Oidium  albicans.     (Kohle  and  Wassermann.) 

mouth  and  throat  in  young  animals  only,  producing  vesicles,  then  white 
membranous  patches  composed  of  the  mycelium  of  the  fungus  (Fig.  162). 
It  is  to  be  recognized  in  such  cases  by  microscopical  examination.  The 
same  disease  affects  children  and  is  found  in  fowls,  calves,  and  colts. 

*  Prepared  by  Charles  Thorn. 


MICROBIAL  DISEASES   OF  MAN  AND   DOMESTIC  ANIMALS      777 

Although  this  disease  has  been  more  common  in  the  tropics,  enough 
cases  are  known  from  the  American  mainland  to  indicate  an  increasing 
importance. 

DERMATOMYCOSES* 

The  molds  which  cause  skin  diseases  form  a  small  group,  with  very 
ill-defined  relationships  to  the  commoner  forms  of  fungi.  They  pro- 
duce a  vegetative  mycelium  within  the  tissues  of  the  host  with  fertile 
branches  which  bear  conidia  but  indicate  little  as  to  their  group  rela- 
tionships among  fungi.  Certain  of  these  diseases  have  been  carefully 
studied,  mostly  from  the  pathological  side,  but  a  very  large  number  of 
such  lesions  are  recorded  without  adequate  study  of  the  organs  involved. 
Most  of  these  diseases  are  tropical  but  considerable  numbers  of  cases 
have  been  recognized  in  temperate  America  in  recent  years.  A  few 
of  these  diseases  are  practically  cosmopolitan. 


FIG.  163. — Trichophylon  tonsurans.     (After  Hyde,  from  Adami  and  Nicholls.) 

BARBER'S  ITCH,  RINGWORM,  HERPES  TONSURANS,  TRICHOMYCOSIS. 
The  disease  due  to  Trichophyton  tonsurans  (Fig.  163),  Malm,  has  re- 
ceived many  names  in  different  languages.  It  attacks  man  and  dom- 
estic animals,  the  ox,  horse,  dog,  cat,  sheep,  hog,  probably  other  animals 

•  Prepared  by  Charles  Thorn. 


778   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

as  well.  It  is  characterized  by  the  formation  of  circular  patches  from 
which  eventually  the  hairs  fall.  These  patches  enlarge  radially  and  fuse 
into  large  areas  covered  with  crusts  with  more  or  less  discharge  in  the 
center.  The  fungus  is  recognized,  microscopically  by  examination 
of  hairs  pulled  from  the  growing  edge  of  the  infection.  The  hyphae 
penetrate  the  layers  of  the  skin  and  especially  surround  the  roots  of 
the  hairs  which,  when  first  affected,  stand  stiff  and  straight. 

The  appearances  of  the  disease  differ  in  the  various  species  of  in- 
fected animals,  as  also  does  the  length  of  time  it  continues.  The 
disease  does  not  affect  the  general  health  greatly,  since  it  primarily 
attacks  the  drier  and  more  horn-like  portions  of  the  skin,  but  becomes 
conspicuous  by  the  falling  of  the  hair  and  by  the  scabs  or  crusts  with 
accompanying  itching  and  discomfort.  Other  species  of  the  same  genus 
have  been  described  which  produce  infected  areas  differing  in  detail 
but  similar  in  their  general  characters. 

FAVUS. — Favus  is  caused  by  Achorion  schonleinii,  Remak,  and 
affects  man,  cats,  dogs,  mice,  rabbits,  and  fowls,  and  many  wild  animals. 
This  is  characterized  by  crusts,  thickened  at  the  edges  and  somewhat 
cup-shaped  in  center,  composed  of  the  mycelium  of  the  mold  cemented 
together  into  masses  by  glairy  substance.  Below,  these  crusts  are  in 
contact  with  the  true  skin.  The  fungus  penetrates  especially  into  the 
hair-follicles  and  hairs  themselves,  which  later  are  shed.  It  attacks 
different  species  of  animals  with  varying  symptoms,  but  produces  more 
serious  lesions  than  those  of  Trichophyton.  Favus  is  especially  serious 
as  it  attacks  man.  Efforts  to  show  that  this  fungus  is  merely  a  parasitic 
form  of  some  species  of  higher  fungi  have  failed.  The  diseased  con- 
ditions have  become  so  well  defined  and  are  reproduced  so  uniformly 
as  to  indicate  a  fixed  habit  in  the  organisms,  whatever  its  source  or 
relationship. 

ACTINOMYCOSIS* 

Actinomyces  bovis\ 

This-  is  a  rather  common  disease  of  domestic  animals,  especially 
cattle.  It  prevails  in  Europe,  North  and  South  America,  and  is  known 
by  various  names  as  lumpy  jaw  and  wooden  tongue.  Cattle  are  most 

*  Prepared  by  M.  H.  Reynolds. 

^Actinomyces  bovis  has  been  classified  by  Frost  (page  in)  as  a  species  of  bacteria,  but. 
yeaause  of  many  features,  it  is  here  inserted  with  the  organisms  strictly  belonging  to  molds  and 
yeasts.— Ed. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      779 

commonly  affected,  but  humans,  hogs,  horses,  sheep,  and  dogs  are  sus- 
ceptible. Actinomyces  produces  a  local  disease  which  never  spreads 
widely  or  rapidly. 

Actinomycosis  is  to  be  considered  as  an  infectious  disease  which 
spreads  by  inoculation. 

The  disease  produced  by  this  microorganism  usually  runs  a  chronic 
course  and  is  distinguished  especially  by  enlargement  of  affected  parts, 
by  hardening  of  the  tongue,  and  by  suppuration.  The  latter  is  one  of 
the  most  constant  and  conspicuous  characteristics.  Head  parts,  in- 
cluding the  facial  bones,  are  commonly  affected;  lungs  and  various  other 
internal  organs  and  even  the  vertebrae  may  be  involved. 

The  extent  of  injury  done  by  this  fungus  depends  on  the  location 
and  size  of  the  involved  area.  Usually  the  most  conspicuous  injury  is 
impaired  nutrition. 


FIG.   164. — Actinomyces     bovis.     The  •  ray-fungus     from     cow.     (Diagrammatic.) 

(After  Williams.) 

There  is  probably  but  little  risk  to  human  health  from  actinomycosis 
n  cattle  as  parts  of  the  carcass  most  commonly  affected  are  not  eaten 
and  edible  parts  are  usually  cooked.  It  is  generally  considered  that 
sound  portions  of  carcasses  which  do  not  show  generalized  disease  are 
fit  for  human  food  purposes. 

There  are  apparently  several  varieties  of  Actinomyces  all  of  which 
are  recognized  for  the  present  as  Actinomyces  bovis. 

The  varieties  of  Actinomyces  are  to  be  regarded  as  members  of  a  very 
complicated  group  of  microorganisms  higher  than  bacteria  and  are 
generallv  spoken  of  as  fungi.  Actinomyces  boms  is  commonly  known 


780   MICROBIOLOGY   OF  DISEASES   OF  MAN  AND   DOMESTIC   ANIMALS 

as  the  ray-fungus  (Fig.  164).  Its  relation  to  the  disease  of  actinomycosis 
is  probably  specific  but  it  is  frequently  aided  by  pus  producing  bacteria. 
It  is  believed  that  the  Actinomyces  vegetate  on  various  grasses, 
especially  wild  barley,  and  that  infection  occurs  by  inoculation  with  the 
awns  and  barbs  of  such  grasses  through  the  mucous  membrane  of  the 
mouth  or  other  portions  of  the  alimentary  tract. 


FIG.  i6«;. — Actinomycosis.     Actinomyces  bovis.     Preparation  from  a  pure  culture. 
Xiooo.     (After  Williams.) 

Infection  by  inoculation  is  the  most  common  method  of  introducing 
the  disease;  but  infection  by  inhalation  evidently  occurs  in  some  cases. 
It  seems  probable  that  some  special  stage  of  development  for  the  Acti- 
nomyces is  necessary  either  within  the  diseased  animal  body  or  upon 
some  plants,  in  order  that  it  may  be  able  to  infect  animal  bodies,  for 
direct  inoculation  by  pus  has  usually  given  negative  results.  Inocula- 
tion by  bits  of  diseased  tissue  occasionally  gives  positive  results. 

It  is  evidently  not  a  producer  of  active  toxins  for  the  disease  dis- 
turbances are  apparently  due  to  harmful  growth  in  the  tissues  and  to 
secondary  infection. 

Suppuration  is  one  of  the  conspicuous  features  as  is  also  the  develop- 
ment of  much  new  granulation  tissue  which  tends  to  degenerate  at  the 
center.  Soft  organs  affected  by  this  parasite  show  a  tendency  to  multi- 
ple abscesses. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      781 

Actinomyces  bovis  grows  rapidly  on  a  variety  of  laboratory  media.  On  glycerin 
agar  the  colonies  develop  into  transparent  drop-like  bodies  in  four  or  five  days  at 
37°.  Old  colonies  become  white  or  yellowish  with  a  powdery  surface.  The  cultural 
and  other  peculiarities  vary  much  and  according  to  the  variety  under  observation. 
Some  varieties  appear  distinctly  aerobic  and  others  anaerobic.  As  a  rule  it  liquefies 
gelatin  growing  in  spherical  masses  which  settle  to  the  bottom  of  the  liquid.  Fila- 
ments appear  in  artificial  growth  which  are  very  long  and  slender,  and  about  5/1 
in  diameter,  and  show  true  branching  (Fig.  165).  The  young  colony  is  a  loose 
mass  of  filaments;  older  colonies  become  dense  and  felted.  Rod-shaped  and  spherical 
forms  appear  in  artificial  cultures.  Cultures,  especially  those  containing  the  round 
forms,  are  very  resistant  to  heat,  light,  drying  and  disinfectants.  Stains  easily. 
Tissue  section  stained  with  carmine  followed  by  Gram's  method  gives  good  results, 
the  thread  showing  dark  and  clubs  red.  Carmine  followed  by  Weigert  gives  a  beau- 
tiful stain.  May  be  recognized  as  visible  granules  found  floating  in  the  pus  in  case 
of  suppuration,  or  embedded  in  tissue.  These  granules  vary  in  color;  some  are  clear 
or  yellow;  others  are  quite  dark.  The  colony  as  it  appears  in  tissue  section  or  pus 
smear  consists  of  a  rosette  arrangement.  The  central  portion  of  the  colony  is  a  dense 
mass  of  mycelium  and  spherical  bodies.  From  this  felted  central  mass,  there  extend 
rays  or  club-like  bodies.  Club-shaped  enlargements  at  the  ends  of  filaments  fre- 
quently appear  and  are  regarded  as  a  distinguishing  characteristic  of  Actinomyces, 
This  organism  is  usually  destroyed  at  75°  for  thirty  minutes.  Final  diagnosis  must 
rest  upon  actual  demonstration  under  the  microscope  which  is  not  difficult.  The 
granular  masses  may  be  washed  in  normal  salt  solution;  and  examined  unstained, 
or  stained  in  diluted  carbol  fuchsin. 

Escape  from  the  diseased  body  is  usually  in  pus  discharged  from 
actinomycotic  abscesses.  In  case  of  open  lung  or  intestinal  lesions 
it  may  be  discharged  through  the  trachea  or  intestines. 

Actinomycotic  pus  scattered  over  fodder,  mangers,  and  feed  racks 
probably  serves  indirectly  as  a  source  of  dissemination. 

Actinomycosis  is  not  a  disease  of  rapid  or  extensive  dissemination. 
Control  work  is  usually  confined  to  isolation,  to  proper  disposition 
of  diseased  animals  and  to  suitable  disinfection. 

ACTINOBACILLOSIS. — Actinobacillosis  is  probably  to  be  distinguished 
from  actinomycosis.  It  is  very  similar  in  subjects  affected,  in  history 
and  clinical  evidence,  but  apparently  different  as  to  specific  cause. 
The  cause  of  actinobacillosis  seems  to  be  a  very  small  bacterium  found 
also  in  rosette- like  masses  resembling  those  of  Actinomyces. 

MYCETOMA  (MADURA  FOOT)* 

This  disease  is  endemic  in  India,  especially  in  Madura,  and  is  found 
in  other  warm  countries. 

•  Prepared  by  Edward  Pidlar, 


782    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

It  is  a  chronic  inflammatory  process  found  most  commonly  in  the 
foot,  occasionally  in  the  hand  but  very  rarely  elsewhere.  It  is  charac- 
terized by  swelling  and  irregular  deformities  of  the  part  with  the 
occurrence  of  sinuses  whence  there  is  a  purulent  discharge  containing 
granules  suggesting  those  of  actinomycosis.  These  granules  may  be 
whitish,  yellowish,  reddish,  or  black  in  color. 

The  causative  organism  is  generally  regarded  as  a  fungus.  It 
is  not  unlikely  that  some  cases  of  the  disease  may  be  confused  with 
actinomycosis.  Several  different  molds  have  been  described,  some  of 
which  have  been  classed  as  Aspergilli,  while  others  have  been  given  new 
names.  It  is  probable  that,  while  the  disease  is  a  fairly  well-marked 
clinical  entity,  the  etiological  agent  varies  in  different  localities. 

Successful  inoculation  of  the  monkey  with  the  white  variety  and  of 
pigeons  with  the  black  variety  has  been  recorded. 

MYCOTIC  LYMPHANGITIS* 
Saccharomyces  jarciminosus\ 

The  disease  caused  by  this  yeast-like  fungus  has  been  called  Japa- 
nese farcy,  epizootic  lymphangitis,  and  mycotic  lymphangitis.  This 
disease  was  first  recognized  in  the  United  States  in  1907.  It  has  already 
been  found  in  Pennsylvania,  Iowa,  California,  and  North  Dakota. 

Saccharomyces  farciminosus  produces  a  slow,  chronic,  contagious 
disease  of  horses  and  mules.  Cattle  appear  susceptible  but  rarely 
show  clinical  symptoms  of  infection. 

This  Saccharomyces  involves  especially  the  superficial  lymphatic 
vessels  and  glands,  but  internal  organs  are  occasionally  affected. 
The  disease  is  essentially  local,  constitutional  disturbances  being  slight. 
The  disease  produced  is  fatal  in  about  10  to  15  per  cent  of  cases  affected 
but  is  much  more  serious  than  these  figures  would  indicate.  Other 
horses  that  do  not  die  are  rendered  useless  for  service,  the  sale  value 
being  ruined  in  many  cases. 

The  lesions  produced  by  this  parasite  resemble  most  closely  the 
farcy  form  of  glanders  but  may  be  easily  distinguished  by  quite  different 
ulcers.  The  pus  is  thick,  creamy,  and  usually  yellow,  whereas  the  pus 

*  Prepared  by  M.  H.  Reynolds. 

t  Work  done  by  Paige,  Frothingham  and  Paige,  Meyer  and  others  raises  questions  concern- 
ing specific  etiology  and  proper  classification,  but  it  is  deemed  wise  to  continue  this  recognition 
and  classification  for  the  present.  Various  authorities  classify  thj§  organism  as  cryptococcus, 
blastomyces,  etc, 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      783 

• 

from  the  farcy  buds  is  clear  and  viscid.  Farcy  cases  respond  to  the 
mallein  test;  lymphangitis  cases  do  not. 

It  seems  to  have  been  well  established  that  Saccharomyces  farci- 
minosus  is  the  direct  cause  of  mycotic  lymphangitis — at  least  of  one 
form  of  it. 

The  Saccharomyces  grows  in  the  animal  tissues  and  by  its  presence 
and  products  acts  as  a  direct  exciting  cause  of  the  disease.  Entrance 
is  effected  through  wounds  which  may  be  very  superficial  and  very 
trivial,  most  frequently  perhaps  on  the  legs,  shoulders,  and  neck. 
The  incubation  period  varies  from  a  few  weeks  to  several  months. 

This  Saccharomyces  is  distributed  through  lymph  vessels,  chiefly 
superficial  ones,  the  nodules  appearing  first  near  the  point  of 
inoculation. 

The  tissue  changes  produced  are  infection,  inflammation,  and 
suppuration  of  the  lymph  vessels  and  glands.  At  first  the  lymph 
vessels  enlarge  and  harden;  then  nodules  develop  under  the  skin  along 
the  course  of  the  vessels.  These  nodules  suppurate  and  the  small 
abscess  cavity  fills  up  with  bright  red  granulation  tissue.  An  entire 
limb  may  enlarge  very  greatly  by  reason  of  excessive  connective- 
tissue  formation,  and  the  greatly  thickened  skin. 

Saccharomyces  farciminosus  is  a  yeast-like  fungus,  ovoid  in  shape  and  3^1  to  5^ 
long  by  2.5/1  to  3.5^1  broad.  This  fungus  grows  slowly  under  artificial  conditions  on 
agar  and  bouillon  after  inoculation  with  pus  from  an  abscess.  It  reproduces  by 
budding  and  does  not  stain  well  by  common  laboratory  stains.  Claudius'  method 
of  staining  gives  good  results. 

Cases  should  be  isolated  and  stables  disinfected  by  the  free  use  of 
very  strong  disinfectants  as  this  Saccharomyces  is  not  easily  killed  by 
ordinary  disinfecting  solutions. 

Another  mycotic  organism  has  more  recently  been  reported* 
in  the  United  States  as  causing  a  lymphangitis  very  similar  clinically 
to  the  lymphangitis  caused  by  Saccharomyces  farciminosus.  Cases 
supposed  to  have  been  plain  cases  of  the  Saccharomyces  form  showed 
on  laboratory  examination  a  Sporothrix  acting  as  the  direct  cause. 
These  workers*  reproduced  cases  by  inoculation  and  recovered  an 
organism  differing  very  materially  from  Saccharomyces  farciminosus. 

*  Sporothrix  and  Epizootic  Lymphangitis,  Paige,  Prothingham,  and  Paige.  Journal  of 
Medical  Research,  Vol.  XXIII,  No.  i.  This  has  been  previously  reported  by  Shenck,  Hek- 
toen,  and  others  for  the  human. 


784   MICROBIOLOGY   OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

The  case  history  and  lesions  produced  parallel  very  closely  those  pro- 
duced by  the  Saccharomyces.  This  Sporothrix  seems  to  have  great 
vitality,  remaining  virulent  in  dried  pus  at  a  temperature  of  —7°  for 
three  months  or  more. 

The  same  organism  has  been  recovered  from  similar  lesions  of  the 
human  where  it  was  apparently  acting  as  the  direct  exciting  cause. 
If  this  be  confirmed,  we  have  two  apparently  different  organisms  cap- 
able of  producing  a  similar  mycotic  lymphangitis.  Complement 
fixation  work  by  Meyer  suggests  that  the  two  may  be  at  least  more 
closely  related  than  was  previously  supposed. 

DISEASES  CAUSED  BY  BACTERIA* 

BOTRYOMYCOSIsf 

Micrococcus  pyogenes 

We  have  typically  in  this  disease  closed  abscesses  with  very  tough 
fibrous  walls  and  slow  development.  These  abscesses  involve  espe- 
cially subcutaneous  and  intermuscular  connective  tissue,  although 
typical  lesions  have  been  found  in  various  internal  organs. 

This  affection  is  probably  limited  to  equines.  The  essential  char- 
acteristic of  this  disease  is  the  presence  of  the  peculiar  masses  of  micro- 
cocci  (Bellinger's  granules).  This  massing  seems  to  occur  only  in 
chronic  cases  where  a  certain  degree  of  immunity  has  developed. 

The  identity  and  proper  classification  of  a  specific  microorganism 
is  still  in  dispute.  Johne  found  M.  ascoformans  acting  as  an  etiolog- 
ical  factor.  Kitt  and  others  found  micrococci  which  could  not  be  dis- 
tinguished from  M.  pyogenes.  Moore  found  a  variety  of  pyogenic 
micrococci  and  streptococci  apparently  serving  as  causative  agents 
and  reports  one  case  of  an  enlarged  spermatic  cord  where  he  found  a 
fungus  resembling  Actinomyces  boms.  Others  identify  Botryomyces 
equi  as  Staphylococcus  pyogenes  aureus,  etc. 

Primary  infection  occurs  by  inoculation  and  not  infrequently 
follows  surgical  operations,  e.g.,  castration.  The  primary  infection 
may  then  lead  to  involvement  of  internal  organs  by  metastasis.  The 
local  effect  here  is  that  of  an  irritant  and  both  irritant  and  tissue 
response  appear  to  resemble  those  that  occur  in  actinomycosis. 

*  Arranged  alphabetically  under  each  of   the  following  families:  Coccacece  (Micrococcus, 
Streptococcus),  Bacteriacece  (Bacterium,  Bacillus,  Pseudomonas),  Spirillacece  (Microspira). 
t  Prepared  by  M.  H.  Reynolds. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      785 

Botryomycosis  is  easily  distinguished  from  actinomycosis  on 
microscopic  examination.  Cases  that  resemble  the  farcy  form  of  glan- 
ders are  easily  distinguished  by  mallein  test,  by  laboratory  animal  in- 
oculation and  by  lack  of  adjacent  lymph-gland  involvement. 

GONORRHCEA* 

Micrococcus   gonorrhoea 

Gonorrhoea  is  one  of  the  most  prevalent  of  the  bacterial  diseases  and 
is  found  throughout  the  civilized  world  and  is  confined  to  the  human 
race. 

The  urogenital  tract  is  the  most  frequent  seat  of  infection  but 
orchitis,  conjunctivitis,  and  arthritis  are  not  uncommon  and  endo- 
carditis and  .a  septicaemic  condition  may  also  occur.  Ophthalmia 
neonatorum  is  due  to  this  organism.  The  ordinary  infections  of  the 
urogenital  tract  have  an  incubation  period  of  from  two  to  eight  days. 
The  inflamed  mucous  membranes  give  rise  to  more  or  less  pain  and  yield 
a  thick  yellow  discharge. 


FIG.  166. — Gonococci  and  pus  cells.     X  1000.     (After  Williams.} 

While  the  fatality  due  directly  to  Gonococcus  infection  is  not  high, 
the  frequent  tendency  to  chronicity  and  its  connection  with  blindness 
id  sterility  render  it  one  of  the  most  important  diseases. 


Prepared  by  Edward  Fidlar. 
50 


786   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

Gonorrhoea  has  been  known  from  the  very  earliest  times.  In  1879 
the  diplococcus  was  pointed  out  by  Neisser  as  the  probable  cause. 
Bumm  in  1885  first  cultivated  it  on  coagulated  human  placental  serum. 

The  microorganisms  can  be  easily  stained  in  the  typical  early  dis- 
charges where  it  occurs  in  pairs  and  for  the  most  part  within  cells 
(Fig.  166). 

For  isolation,  sterile  human  blood,  blood-serum  or  ascitic  fluid  should  be  added 
to  melted  nutrient  agar  at  about  45°.  Thompson's  plasma  glucose  agar  and  Wasser- 
mann's  swine-serum-nutrose  medium  are  also  good.  The  Gonococcus  is  about  o.6ju 
to  o.S/x  in  diameter.  It  is  usually  seen  in  pairs;  where  the  adjacent  sides  of  the 
cocci  are  flattened  the  long  diameter  of  the  pair  reaches  as  much  as  i.6/x;  non-motile, 
and  forms  neither  spores  nor  capsules.  It  stains  readily  with  the  aniline  dyes  and 
is  Gram-negative.  The  temperature  range  is  30°  to  38.5°  with  an  optimum  of  37.5°. 
Aerobic  conditions  are  preferred  with  a  reaction  about  0.6  per  cent,  acid  to  phenol- 
phthalein,  but  Ruediger  reports  luxuriant  growth  in  air-tight-tubes  of  special  neu- 
tral serum  agar.  On  serum  agar  or  similar  suitable  media,  colonies  appear  in 
twenty-four  hours  as  fine  slightly  elevated,  translucent  or  opalescent  spots  frequently 
referred  to  as  "dew-drop"  colonies.  They  possess  a  faint  bluish  or  grayish  white 
color  with  a  slightly  marked  concentric  or  radial  striation  with  a  scalloped  margin 
and  finely  granular  center.  In  serum  broth  there  may  occasionally  be  a  uniform 
clouding  though,  as  a  rule,  there  is  a  finely  granular,  somewhat  slimy  sediment  with 
clear  fluid  above.  Only  in  exceptional  cases  has  growth  been  observed  in  gelatin 
because  of  the  unfavorable  temperature.  On  inspissated  blood  serum  growth  may 
sometimes  be  observed  as  discrete  pale  yellowish  or  brownish  colonies.  Dextrose 
is  changed  with  the  development  of  acid  but  no  gas.  Alkali  is  not  formed  in  any 
medium  by  typical  strains.  No  gas,  indol  or  pigment  are  formed.  The  toxins  are 
intracellular  and  quite  thermostable.  Resistance  is  very  slight  toward  external 
influences.  Cultures  undergo  rapid  autolytic  changes  and  die  out  at  room  tempera- 
ture, often  within  forty-eight  hours.  A  temperature  of  41°  to  45°  will  kill  in  a  few 
hours.  To  light  and  drying  they  are  also  very  sensitive,  and  are  rapidly  killed  by 
the  ordinary  disinfectants. 

Animals  inoculated  subcutaneously  or  intraperitoneally  show  symp- 
toms of  poisoning  with  suppuration  and  necrosis  locally  and  may 
succumb. 

The  virulence  of  the  organism  is  variable.  They  may  apparently  lie 
dormant  or  rather,  may  remain  very  slightly  active  in  chronic  conditions 
in  one  individual  but  set  up  an  acute  gonorrhoea  when  transferred 
to  a  second  person. 

The  organism  gains  entrance  to  the  urethral  mucosa  or  conjunctiva 
usually  by  direct  contact  and  it  is  doubtful  if  the  disease  could  be 
carried  by  any  infected  article  later  than  twenty-four  hours. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      787 

The  organism  is  found  at  the  local  lesion  and  has  been  obtained 
from  the  fluid  of  affected  joints,  and  from  the  blood  in  the  septicaemic 
cases. 

A  general  immunity  is  seldom  if  ever  developed  in  man  following  an 
attack  of  gonorrhoea.  A  complement-fixing  antibody  has  been  dem- 
onstrated, however,  which  is  sometimes  an  aid  in  diagnosis  when  a  good 
polyvalent  antigen  is  used  in  the  test.  Thomson's  antigen  is  an 
improvement. 

Injections  of  cultures  into  animals  give  rise  to  agglutinins,  bacteri- 
cidal and  complement-binding  bodies. 

The  diplococcus  is  eliminated  only  in  the  purulent  discharge. 

Great  importance  attaches  to  the  fact  that  persons  may  harbor  the 
Gonococcus  long  after  the  acute  condition  has  disappeared  and  when  the 
coccus  seems  to  be  no  longer  harmful  to  its  host.  Such  cases  bring 
about  untold  misery  and  form  one  of  the  most  difficult  problems  in  both 
medical  and  social  science.  It  has  been  stated  that  Gonococci  have 
been  found  as  late  as  twenty  years  after  the  primary  infection. 

The  most  extended  and  successful  prophylactic  measures  have  been 
carried  out  in  the  armies  and  navies  of  various  countries  by  the  use  of 
germicidal  solutions  whenever  there  has  been  any  chance  of  exposure 
to  infection.  The  use  of  germicidal  solutions  in  the  eyes  of  new- 
born infants  is  practically  universal  as  a  preventive  measure  against 
ophthalmia. 

EPIDEMIC  CEREBRO-SPINAL  MENINGITIS* 
Micrococcus  meningitidis 

Cerebro-spinal  meningitis  may  be  caused  by  different  bacteria  such 
as  the  pneumococcus,  streptococcus,  staphylococcus,  influenza  bacillus, 
tubercle  bacillus,  etc.,  but  the  greater  proportion  of  cases  of  acute 
meningitis,  those  of  the  epidemic  type,  are  due  to  the  meningococcus 
or  diplococcus  intracellularis  meningitidis. 

Epidemic  meningitis  has  been  described  chiefly  in  Europe  and  Amer- 
ica and  appears  to  have  been  first  clearly  defined  in  1805.  While 
sporadic  cases  occur,  the  disease  usually  exists  in  the  epidemic  form, 
beginning  in  the  fall,  continuing  during  the  winter,  and  declining  in  the 
spring.  Of  late  years  it  would  seem  to  be  on  the  increase. 

*  Prepared  by  Edward  Fidlar. 


788   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

The  incubation  period  is  unknown. 

There  is  considerable  variety  in  the  character  of  the  cases.  As  a 
rule  the  invasion  is  sudden  with  headache  and  vomiting  as  prominent 
symptoms.  The  headache  usually  increases,  with  disturbances  of 
vision,  restlessness,  and  pains  and  rigidity  in  the  muscles  of  the  back 
and  neck.  The  temperature  is  irregular  and  variable,  the  usual  being 
about  101°  to  102°.  Herpes  occurs  frequently  and  a  purpuric  rash  is 
common,  especially  in  the  severe  cases,  so  that  the  term  "spotted  fever" 
has  sometimes  been  given  to  the  disease.  The  patient  usually  passes 
into  a  stuporous  state,  though  delirium  may  occur  before  it.  Death 
may  occur  in  a  few  hours  (fulminant  type)  or  within  a  week,  or  occa- 
sionally may  be  postponed  as  late  as  six  months.  In  all  favorable 
cases  the  recovery  is  slow. 

A  fibrinous  exudate  which  occurs  chiefly  at  the  base  of  the  brain, 
and  the  presence  of  pus  cells  in  the  cerebro-spinal  fluid,  are  prominent 
pathological  features,  but  in  the  fulminant  cases  the  gross  pathological 
findings  may  be  surprisingly  insignificant. 

According  to  Flexner,  serum  treatment  has  reduced  the  mortality 
of  the  disease  from  about  70  per  cent  to  30  per  cent,  and  to  less  than 
20  per  cent  if  treatment  is  begun  within  the  first  three  days. 

The  demonstration  of  the  organism  in  the  cerebro-spinal  fluid  of 
the  typical  case  may  sometimes  be  an  easy  matter,  but  at  other  times 
may  require  a  prolonged  search.  It  appears  as  a  Gram-negative  coccus, 
single  and  in  pairs,  frequently  within  pus  cells  but  occasionally  extra- 
cellular. From  the  usual  case  it  can  be  obtained  in  pure  culture  by 
sowing  the  sediment  from  the  spinal  fluid  upon  suitable  media.  The 
amount  of  material  planted  should  be  abundant  and  to  supplement 
these  first  cultures  it  is  well  to  incubate  the  fluid  at  37°  for  twelve  to 
eighteen  hours  and  then  make  further  inoculations.  The  media  used 
are  often  blood  agar  or  serum  glucose  agar,  but  legumin-trypagar  was 
most  successful  in  the  British  army  during  the  war. 

As  found  in  culture  media  the  meningococcus  will  show  swollen 
involution  forms  often  in  comparatively  young  cultures.  There  are  no 
spores,  flagella  nor  capsules.  It  can  be  stained  readily  by  the  aniline 
dyes  and  with  methylene  blue  will  sometimes  show  metachromatism. 
It  is  Gram-negative. 

The  temperature  relations  are  of  some  importance  in  identifying 
the  coccus.  It  has  a  minimum  temperature  of  about  25°,  an  opti- 


MICROBIAL  DISEASES   OF   MAN  AND   DOMESTIC   ANIMALS      789 

mum  of  37°  and  a  maximum  of  42°.     Its  atmospheric  requirements  are 
those  of  an  aerobe. 

On  serum  agar  the  colonies  are  small,  grayish  and  glistening,  with 
smooth  outline  and  granular  center.  On  legumin-agar  they  are 
round,  hemispherically  raised,  and  opalescent.  In  broth  growth  is 
slow  and  occurs  at  the  surface.  Only  rarely  is  growth  obtained  on 
gelatin  media  chiefly  because  of  the  unfavorable  temperature  required. 
There  is  no  change  in  litmus  milk.  Acid  is  formed  from  dextrose  and 
maltose. 

The  toxins  of  the  meningococcus  are  probably  intracellular. 

The  resistance  of  the  organism  to  unfavorable  conditions  is  very 
slight,  and  it  undergoes  autolytic  changes  almost  with  the  same  rapidity 
as  does  the  gonococcus. 

Meningitis  due  to  this  coccus  does  not  occur  naturally  in  animals, 
but  it  has  been  produced  in  monkeys  artificially.  Laboratory  animals 
inoculated  subcutaneously,  intraperitoneally  or  intravenously  with  a 
sufficiently  large  dose  will  die  without  developing  meningitis. 

Animals  immunized  by  graded  doses  show  specific  agglutinins, 
opsonins  and  lysins.  Horses  so  treated  yield  a  serum  which  in  some 
hands  has  given  very  favorable  results.  In  the  first  epidemics  in  the 
British  armies  however,  the  mortality  ranged  from  about  40  to  50  per 
cent  and  the  serum  was  distinctly  disappointing.  Investigation  then 
demonstrated  that  four  types  of  the  meningococcus  existed,  distin-- 
guishable  by  agglutination  reactions,  and  new  sera  were  produced  which 
yielded  better  results. 

As  the  germs  leave  the  body  in  the  discharges  of  the  nose  and  mouth, 
the  prevention  and  control  of  the  disease  would  appear  at  first  thought 
to  be  an  easy  matter,  but  the  occurrence  of  carriers  and  ignorance  of 
the  factors  which  govern  the  virulence  of  the  infective  agent  and  the 
individual's  susceptibility  make  epidemic  meningitis  a  very  difficult 
problem  from  the  standpoint  of  public  health. 

INFECTIOUS  MASTITIS* 

Infectious  mastitis  or  mammitis  (inflammation  of  the  udder) 
appears  in  isolated  outbreaks  and  is  serious  for  the  individual  owner  and 
individual  herd,  but  it  never  spreads  widely.  It  may  affect  a  large 
portion  of  the  herd  and  cause  heavy  financial  losses.  Infectious  masti- 

*  Prepared  by  M.  H.  Reynolds. 


7QO   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

tis  may  have  serious  significance  for  children  and  others  consuming 
milk;  but  there  is  little  information  on  this  point,  based  on  careful 
work. 

This  is  to  be  considered  as  an  infectious,  enzootic  disease  and  prob- 
ably not  specific.  There  is  good  reason  to  suppose  that  different  out- 
breaks have  been  due  to  several  different  pyogenic  or  pus-producing 
organisms. 

We  cannot  consider  any  one  species  of  bacteria  as  the  specific  cause. 
Various  micrococci,  streptococci,  and  staphylococci  have  been  found 
acting  as  causal  agents. 

Recent  evidence  indicates  that  udders  of  apparently  healthy  cows 
may  contain  a  variety  of  bacteria  and  that  the  infections  may  remain 
more  or  less  permanent.  This  is  in  part  the  explanation  of  recurrent 
cases  of  mastitis. 

Discharge  is  either  through  the  teat  or  rarely  by  external  rupture 
of  abscess.  Transmission  from  cow  to  cow  is  indirect,  and  frequently 
on  milkers'  hands. 

Entrance  is  usually  effected  by  way  of  the  milk  ducts,  thence  into 
the  milk  cistern  and  to  more  remote  parts  of  the  gland.  The  infection 
may  also  come  by  way  of  the  blood  or  lymph  channels  to  the  glands. 
A  given  case  may  thus  be  due  to  bacteria  previously  in  the  udder,  the 
attack  being  determined  by  an  area  of  lessened  tissue  resistance  pro- 
duced by  injury. 

In  one  class  of  cases,  the  gland  structures  are  first  involved;  in  other 
cases  the  connective  tissue  frame-work  is  first  involved.  In  one  type  of 
this  disease  caused  by  streptococci  these  microorganisms  attack  espe- 
cially the  mucous  membrane  lining  milk  ducts  and  produce  a  catarrhal 
disease  of  that  membrane.  This  is  indicated  by  a  cord-like  swelling 
which  extends  along  the  milk  canal  through  the  teat  to  the  milk  cistern. 
This  infection  frequently  leads  to  "blind  quarter;"  i.e.,  to  closure  of  the 
teat  canal  and  loss  of  the  quarter;  or  this  infection  may  lead  to  the  for- 
mation of  one  or  more  pea-like  nodules  along  the  teat  canal  and  conse- 
quent obstruction. 

In  many  cases  the  lactose  is  decomposed  by  the  invading  organisms, 
leading  to  the  formation  of  organic  acids.  These  acids  produce  coagu- 
lation. The  coagula  soon  obstruct  the  milk  ducts  and  alveoli  and  the 
secreting  cells  degenerate.  The  invaded  tissues  may  suppurate  or  even 
become  gangrenous. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      7QI 

These  infections  are  indicated  by  dullness,  lack  of  appetite,  fever, 
inflammation  of  the  udder,  and  by  small  nodules  or  cord-like  swelling 
within  and  lengthwise  of  the  teat. 

It  must  be  borne  in  mind  that  the  infecting  microorganism  is  the 
thing  to  be  controlled.  Outbreaks  of  this  disease  frequently  have  origin 
in  infected  cows  added  to  the  herd.  Some  cows  are  unsuspected  "car- 
riers." New  cows  should  be  suspected  until  found  free  by  careful 
examination. 

Affected  cows  should  be  isolated  if  possible,  and  always  milked  last. 
Their  milk  should  be  boiled  and  fed  to  hogs,  and  the  milker's  hands 
suitably  disinfected. 

MALTA  FEVER* 
Micrococcus  melitensis 

This  disease  is  endemic  along  the  shores  of  the  Mediterranean,  in 
South  Africa,  India,  China,  the  Philippines,  and  the  West  Indies. 

The  period  of  incubation  is  usually  about  six  to  ten  days. 

The  ordinary  variety  shows  an  intermittent  or  undulatory  fever 
which  may  be  protracted  to  six  months  or  more,  accompanied  by  consti- 
pation and  general  debility  with  various  complications  such  as  neural- 
gias, arthritis,  orchitis,  etc.  Relapses  occur  after  periods  of  absence  of 
symptoms.  Malignant  cases  are  described  which  may  be  fatal  in  a 
week  or  ten  days.  The  mortality  is  2  per  cent  and  no  characteristic 
pathological  changes  are  found. 

The  etiological  factor  is  M.  melitensis  and  was  described  by  Bruce 
in  1887. 

The  organism  can  be  obtained  from  the  blood  and  in  many  cases 
from  the  urine.  The  most  recently  reported  favorable  medium  for 
blood  cultures  is  peptone  broth  with  the  addition  of  bile. 

It  is  generally  recognized  as  an  oval  coccus,  although  it  is  also  described  as  a 
bacillus.  Its  maximum  measurements  have  been  found  to  be  0.8/1  by  0.53/1,  its 
minimum  diameters  0.55/1  by  0.4/1.  It  occurs  singly,  in  pairs,  in  irregular  groups  and 
in  short  chains.  (Recently  the  organism  has  been  described  as  motile  and  possessing 
a  single  flagellum  at  the  extremity  of  the  long  diameter  of  the  oval  coccus.)  It 
stains  by  ordinary  aniline  dyes  and  is  Gram-negative.  It  grows  slowly  at  room 
temperature,  better  at  body  temperature  and  does  not  seem  to  be  markedly  sensitive 
to  acidity  or  alkalinity  of  reaction.  It  grows  aerobically.  On  plain  agar  after 

*  Prepared  by  Edward  Fidlar 


792    MICROBIOLOGY  OF  DISEASES   OF   MAN  AND   DOMESTIC  ANIMALS 

about  forty-eight  hours  small  whitish  to  yellowish  colonies  appear.  Growth  has 
been  observeed  inbroth  in  eighteen  to  twenty-four  hours,  on  gelatin  in  eight  or  nine 
days,  and  the  latter  is  not  liquefied.  It  has  been  found  to  grow  on  acid  potato  and 
in  acid  or  alkaline  urine. 

Human  beings  and  animals  eliminate  the  organisms  in  the  urine,  and 
the  milk  of  goats  has  been  found  to  be  a  prolific  source  of  infection. 
With  proper  regulations  in  regard  to  goats'  milk  the  disease  has  been 
greatly  reduced. 

STAPHYLOCOCCIC   INFECTIONS* 

Boils,  Abscesses,  Wounds,  Osteomyelitis,  Pyemia,  Etc. 
Micrococcus  pyogenes  var.  aureus,  etc. 

Infections  of  this  order  are  found  throughout  the  world  and  because 
of  the  association  of  staphylococci  and  streptococci  with  the  large 
majority  of  purulent  inflammations,  these  organisms  are  called  the 
pyogenic  cocci. 

No  specific  disease  is  produced,  but  chiefly  boils,  circumscribed 
abscesses,  infected  wounds,  osteomyelitis,  pyaemia,  etc.  The  symptoms 
alone  will  not  indicate  whether  staphylococci  or  streptococci  are 
present,  but  a  low  grade  of  infection  with  more  pus  and  less  constitu- 
tional disturbance  tends  to  indicate  the  former,  and  staphylococci  tend 
to  pyaemia  rather  than  to  septicaemia. 

Pasteur,  Koch,  Ogston  and  Rosenbach  established  the  importance 
of  these  organisms. 

Staphylococci  in  pus  stain  readily  with  aniline  dyes.  Pure  cultures 
can  be  obtained  by  plating  or  streaking  on  plain  nutrient  agar. 

While  several  different  forms  are  found  in  pathological  conditions, 
the  M.  pyogenes  var.  aureus  is  by  far  the  most  frequent,  and  it  is  de- 
scribed here  as  a  type. 

M.  pyogenes  var.  aureus  is  a  spherical  coccus  about  0.7^1  to  O.Q/X  in  diameter  though 
forms  o.4jLt  to  i.2ju  have  been  noted.  On  solid  media  the  organism  may  be  found 
solitary,  in  pairs,  or  in  rows  of  three  or  four,  but  characteristically  in  irregular  groups 
like  bunches  of  grapes.  In  liquid  media  the  single  and  paired  arrangement  is  most 
frequent.  No  spores,  no  capsules  and  no  flagella  are  found;  the  organism  shows 
marked  Brownian  movement,  like  other  cocci;  Gram-positive.  The  temperature 
range  of  growth  is  from  about  10°  to  43°  with  an  optimum  about  30°.  Aerobeand 

•  Prepared  by  Edward  Fidlar. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      793 

facultative  anaerobe.  It  grows  readily  on  all  routine  media,  preferring  a  reaction 
slightly  alkaline  to  litmus.  Growth  on  plain  agar  is  rapid  and  abundant.  After 
twenty-four  hours  there  appear  round  grayish-white  or  yellowish  colonies  about  2 
mm.  in  diameter,  smooth  and  raised  above  the  surface  of  the  medium.  Micro- 
scopically, the  colonies  are  regular  in  outline  and  finely  granular.  The  char- 
acteristic orange-yellow  pigment  may  not  appear  until  later  or  if  already  present  in 
twenty-four  hours,  deepens  with  further  growth.  In  broth,  growth  is  also  rapid 
and  causes  a  diffuse  clouding  with  a  thin  pellicle  and  a  heavy  sediment  after  several 
days.  In  gelatin,  colonies  are  as  on  agar  and  sink  into  cup-like  depressions  as  the 
medium  is  liquefied.  Liquefaction  is  rapid  with  some  strains  and  slower  with  others, 
and  in  old  cultures  is  of  a  funnel  or  saccate  type.  It  is  due  to  a  thermolabile  ferment- 
like  substance  known  as  gelatinase.  In  milk,  the  staphylococcus  grows  readily 
and  causes  coagulation  sometimes  early  but  usually  in  three  or  four  days'  time. 
On  potato  growth  is  usually  abundant;  it  is  not  as  moist  nor  as  smooth  as  on  agar 
and  is  slower.  .  Pigment  is  developed  usually  to  the  highest  degree  and  sometimes 
cultures  appearing  white  on  agar  develop  pigment  on  potato.  On  inspissated 
blood  serum  growth  is  usually  moist  and  abundant.  Occasionally  the  growth 
sinks  slightly  into  the  medium  suggesting  partial  liquefaction.  In  dextrose,  lactose 
and  saccharose  media,  acid  is  produced,  but  no  gas.  Acid  is  a  constant  product. 
Formic,  lactic,  butyric  and  valerianic  acids  have  been  found  and  probably  other 
fatty  acids  occur.  Some  authorities  state  that  indol  is  formed  but  negative  results 
are  the  rule.  Nitrites  are  formed  by  the  reduction  of  nitrates.  A  characteristic 
odor  from  cultures  is  due  probably  to  the  presence  of  fatty  acids.  The  pigment 
appears  in  aerobic  cultures  and  is  absent  in  anaerobic  cultures.  It  is  insoluble  in 
water  but  soluble  in  alcohol,  chloroform,  ether  and  benzol.  The  toxins  are  largely 
intracellular.  A  thermolabile,  haemolytic  substance  may  be  found  in  the  more 
virulent  strains  after  about  ten  days'  growth  in  moderately  alkaline  broth  and  can 
be  freed  by  filtration  through  porcelain  filters.  Another  soluble  toxic  substance  is 
found,  causing  the  death  of  leucocytes — leucocidin.  It  is  considerably  less  stable 
than  the  staphylo-hamolysin.  The  staphylococci  are  among  the  most  resistant  of 
the  non-spore  bearing  bacteria.  Sometimes  60°  for  a  full  hour  or  even  longer  is 
necessary  to  kill  watery  suspensions;  70°  is  usually  necessary  to  kill  in  ten  minutes. 
If  organic  material  is  present  the  resistance  is,  of  course,  much  greater.  Low  tem- 
peratures have  little  effect  and  it  has  been  stated  that  30  per  cent  have  survived 
thirty  minutes'  exposure  to  liquid  air.  To  direct  sunlight  and  drying  staphylococci 
also  show  considerable  resistance  and  may  be  found  in  dried  pus  for  several  months. 
Resistance  to  germicides  is  also  somewhat  greater  than  that  of  other  vegetative 
bacteria,  and  is  increased  especially  in  the  presence  of  organic  material.  In  watery 
suspensions  staphylococci  are  killed  by  i  :  1000  mercuric  chloride  in  ten  to  fifteen 
minutes,  by  3  per  cent  carbolic  acid  in  two  to  ten  minutes  and  by  5  per  cent  formal- 
dehyde in  the  same  time. 


Man  seems  to  be  considerably  more  susceptible  to  staphylococcic  in- 
fections than  animals.  Of  the  latter  rabbits  and  mice  and  guinea-pigs 
are  susceptible  in  this  order. 


794   MICROBIOLOGY  OF  DISEASES   OF   MAN  AND   DOMESTIC  ANIMALS 

The  virulence  of  the  organism  shows  considerable  variation  and  is 
usually  increased  by  successive  passages  through  animals  of  the  same 
species  while  remaining  unaltered  for  animals  of  other  species. 

Subcutaneous  inoculation  usually  results  in  abscess  formation. 
Virulent  cultures  injected  into  the  peritoneal  cavity  of  animals  may  kill 
in  forty-eight  hours  to  a  week  or  even  longer,  with  pysemic  abscesses 
especially  in  the  kidneys.  Malignant  or  ulcerative  endocarditis  has 
been  experimentally  produced  by  intravenous  injection  when  the  heart 
valves  have  been  injured,  chemically  or  mechanically.  Osteomyelitis 
has  also  been  experimentally  produced. 

In  man  simple  rubbing  of  virulent  cultures  into  the  skin  is  often 
sufficient  to  produce  a  furuncle. 

Upon  entering  the  tissue  the  cocci  are  strongly  chemotactic  and  pus 
inevitably  results.  With  virulent  cultures  the  leucocidal  substance  is 
more  or  less  strongly  active.  The  organism  may  be  limited  to  the  first 
abscess  or  by  invasion  of  the  blood  stream  multiple  abscesses  result. 
In  these  cases,  which  are  usually  fatal,  the  organism  will  be  found 
throughout  the  body. 

Immunization  can  be  secured  by  repeated  injections  of  cocci  dead 
or  alive  in  graduated  doses.  The  sera  possess  slight  bactericidal  and 
agglutinating  properties,  and  a  high  degree  of  opsonic  power.  The 
latter  property  is  probably  the  most  important. 

The  serum  of  immunized  animals  is  protective  only  when  used 
slightly  before  or  along  with  the  injection  of  the  organisms  and  is  con- 
sequently of  little  practical  value.  Active  immunization,  however,  is 
being  extensively  practised  particularly  with  the  autogenous  strains. 
Leucocytic  extracts  have  also  been  successfully  though  not  so  widely 
used. 

The  prophylaxis  of  staphylococcic  infections  is  the  same  as  for  other 
pus-producing  forms. 

Several  other  kinds  of  staphylococci  have  been  found  associated  with 
pathological  conditions,  the  most  important  of  which  are  M.  pyogenes 
var.  albus,  M.  epidermidis  albus  (Welch),  and  M.  pyogenes  var.  citreus. 
The  first  seems  to  be  slightly  pathogenic,  and  rarely  produces  severe 
infection.  It  is  distinguished  from  the  aureus  by  lack  of  pigment. 

The  second  variety  appears  to  be  an  attenuated  form  of  the  other. 

The  third  variety  is  distinguished  from  aureus  and  albus  by  the 
development  of  a  lemon-yellow  pigment. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      79$ 

STREPTOCOCCIC  INFECTIONS* 

General  Septiccemia,  Puerperal  Septic&mia,  Erysipelas,  Bronchopneu- 

monia,  Etc. 

Streptococcus  pyogenes 

Several  different  methods  have  been  used  to  classify  streptococci. 
The  species  Strept.  mridans  and  Strept.  hamolyticus,  for  example,  are 
based  on  the  action  upon  haemoglobin.  Fermentation  of  carbohy- 
drates has  claimed  much  attention,  but  no  satisfactory  correlation 
has  been  found  between  a  biochemical  classification  and  the  clinical 
forms  of  infection  encountered.  Work  in  the  British  and  American 
armies  suggests  that  an  immunological  basis  of  division  will  prove  bet- 
ter, and  at  present  four  types  are  indicated.  Some  French  authors 
desire  to  separate  a  group  called  Enterococcus  as  distinct  from,  and  of 
less  pathological  significance  than  Streptococcus. 

Strep tococcic  infections  are  endemic  among  all  races  and  under  all 
social  conditions.  In  the  days  before  antisepsis  and  our  knowledge  of 
the  transmission  of  infectious  diseases,  erysipelas  and  puerperal  sep- 
ticaemia occurred  in  epidemics  that  were  the  scourges  of  surgical  and 
lying-in  hospitals. 

When  the  work  of  Pasteur  and  Lister  became  fully  comprehended 
such  epidemics  ceased  to  exist. 

Natural  streptococcic  infections  have  been  described  in  horses  and 
cattle  and  among  the  laboratory  animals,  but  as  a  rule  such  disease  is 
much  rarer  in  animals  than  in  the  human  being. 

For  septicaemia  and  erysipelas  the  period  of  incubation  is  probably 
from  several  hours  to  three  days.  For  some  conditions  it  is  impossible 
to  determine. 

The  symptoms  of  septicaemia  begin  with  a  rapid  rise  of  temperature 
which  may  reach  io5°F.  or  even  higher.  Chills  accompany  the  fever 
and  are  often  severe.  The  pulse  is  rapid,  irregular  and  weak  and  the 
respiration  labored.  There  may  be  vomiting  and  constipation  or 
diarrhoea.  Headache  is  more  or  less  severe  with  sometimes  delirium. 
In  cases  lasting  for  several  days  the  skin  appears  slightly  jaundiced. 
The  urine  is  of  the  usual  febrile  type  and,  as  a  rule,  shows  the  micro- 
organism causing  the  disease.  Death  may  occur  in  two  or  three  days 
or  within  a  week  or  in  milder  cases  may  be  followed  by  recovery. 

*  Prepared  by  Edward  Fidlar 


796   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

After  death  from  septicaemia  the  body  tends  to  putrefy  rapidly. 
The  glandular  organs  all  tend  to  be  swollen  and  soft,  especially  the 
spleen,  and  parenchymatous  degenerations  are  found  to  a  greater  or 
less  extent.  The  lining  membrane  of  the  heart  and  vessels  is  blood 
stained,  a  rather  characteristic  feature  of  streptococcic  septicaemia. 
Bronchitis  and  broncho-pneumonia  are  usually  found. 

Erysipelas  is  an  inflammation  of  the  skin,  occasionally  of  mucous 
membranes,  and  the  name  is  applied  now  only  when  the  condition  is 
brought  about  by  streptococci.  The  inflamed  area  is  very  definitely 
outlined  and  may  present  blebs  of  a  greater  or  less  size.  (Edema  may 
be  very  marked  where  the  skin  covers  loose  tissue.  Fever  is  present 
with  its  usual  accompaniments.  There  may  be  vomiting,  constipation 
or  diarrhoea.  There  may  be  severe  headaches  or  delirium.  In  fatal 
cases,  death  may  occur  without  any  apparent  complication,  or  it  may 
follow  meningitis,  pericarditis,  nephritis  or  some  other  sequel.  In  sim- 
ple uncomplicated  fatal  cases  the  liver,  kidneys  and  spleen  are  swollen 
and  soft  and  show  degenerative  changes  in  the  gland  cells. 

Bronchopneumonia  may  be  a  primary  condition  or  secondary  to 
some  other  disease.  The  streptococcus  produces  a  purulent  inflam- 
mation in  the  terminal  bronchioles  and  their  surrounding  alveoli. 
This  lobular  distribution  may  become  practically  lobar  by  the  con- 
fluence of  affected  areas.  Streptococcic  bronchopneumonias  are 
frequent  in  fatal  cases  of  influenza. 

Pasteur,  Koch,  Rosenbach  and  Fehleisen  divide  the  earlier  honors  in 
the  gradual  working  out  of  the  relationships  of  streptococci  to  disease. 

Blood  culture  in  plain  broth  in  the  case  of  septicaemia  or  inoculation 
of  plain  nutrient  agar  from  pus  are  practically  always  successful. 
Growth  is  never  luxuriant  on  the  ordinary  media.  Cultivation  from 
cases  of  erysipelas  is  less  easy  because  most  of  the  organisms  are  found 
at  the  margin  of  the  lesion  and  are  difficult  to  reach. 

In  exudates  a  stained  smear  will  usually  demonstrate  the  chain- 
forming  coccus  at  once. 

The  cocci  vary  in  size  from  0.4;*  to  i/x.  In  shape  the  organisms  may  be  rounded 
or  oval  or  with  one  aspect  flattened  when  occurring  in  pairs.  The  chains  may  be  long 
or  short  and  a  grouping  into  pairs  is  frequent  even  within  the  chain.  There  are  no 
true  spores  developed  and  the  organism  is  non-motile.  Capsules  are  not  found  on 
the  majority  of  streptococci.  Staining  the  organism  is  easily  accomplished  with  the 
ordinary  aniline  dyes.  It  is  Gram-positive.  The  temperature  range  in  which 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      797 

streptococci  are  capable  of  growing  is  about  from  15°  to  45°,  the  optimum  tem- 
perature is  about  37°.  Streptococci  are,  as  a  rule,  aerobes  and  facultative  anaerobes. 
Strict  anaerobic  species  are  said  to  have  been  isolated  from  faeces.  The  reaction  of 
media  should  be  slightly  alkaline.  Acid  production  is  a  striking  feature  of  this 
organism  and  has  a  decided  inhibitive  effect  upon  its  growth.  Concerning  the 
action  on  carbohydrates  this  organism  typically  forms  acid  from  monosaccharides, 
lactose,  saccharose,  and  salicin.  Gas  is  never  produced.  Nitrates  are  reduced  by 
some  streptococci  to  nitrites.  The  production  of  hydrogen  sulphide  is  characteristic 
of  some  forms  which  have  been  grouped  as  Strept.  fcecalis.  No  pigment  is  found 
other  than  the  slight  brownish  tinge  seen  in  some  gelatin  cultures.  Typically 
actively  haemolytic.  This  power  may  be  lost  on  cultivation.  The  toxic  products 
of  the  streptococci  have  been  the  subject  of  a  great  deal  of  investigation,  but  few 
definite  facts  have  been  discovered.  When  cultivated  on  plain  nutrient  agar  the 
growth  is  visible  in  eighteen  to  twenty-four  hours  as  small  round  translucent  finely 
granular  colonies,  which  possess  an  even  or  notched  border,  and  a  tendency  to  remain 
discrete  except  when  thickly  sown.  The  center  is  thickened  and  the  margins  thinner. 
In  plain  nutrient  broth  the  majority  of  long-chained  varieties  produce  at  the  bottom 
and  along  the  sides  of  the  tube  a  granular  deposit,  or  small  flocculi  or  large  flakes, 
leaving  the  remainder  of  the  broth  clear.  A  few  long-chained  varieties  cloud  the 
broth  uniformly.  The  short-chained  streptococci  as  a  rule,  produce  a  cloudiness  in 
the  medium  which  remains  for  a  number  of  days  even  though  a  finely  granular 
deposit  accumulates  at  the  bottom  of  the  tube.  On  plates  of  plain  nutrient  gelatin 
the  colony  formation  remains  the  same  as  that  on  agar.  In  stab  cultures  a  finely 
granular  filiform  growth  appears  which  later  may  have  a  beaded  appearance  and 
sometimes  a  brownish  color.  The  gelatin  is  not  liquefied.  Milk  is  a  favorable 
medium  for  the  growth  of  streptococci  and  a  strong  acidity  and  coagulation  some- 
times takes  place.  Growth  on  potato  is  said  not  to  take  place,  but  in  some  cases 
an  invisible  growth  seems  to  occur.  Loeffler's  blood  serum  is  also  a  favorable 
medium.  Streptococci,  as  a  rule,  die  out  rapidly  in  cultures  due  to  the  accumulation 
of  their  own  products.  In  pus,  blood,  sputum,  etc.,  the  organism  may  be  found 
alive  after  several  weeks  or  even  months  at  room  temperature.  The  thermal  death- 
point  is  about  54°  in  ten  minutes.  Direct  sunlight  kills  within  a  few  hours,  and 
they  are  readily  killed  by  many  disinfectants. 

Entrance  of  streptococci  is  afforded  by  any  break  in  the  surface  of 
the  body.  A  local  suppuration  may  be  the  result  or  it  may  be  followed 
by  a  general  septicaemic  condition. 

In  erysipelas  some  local  injury  is  also  probably  necessary  as  a 
starting-point. 

Following  the  local  establishment  of  streptococci  sufficient  toxin 
is  elaborated  to  produce  greater  or  less  systemic  disturbance.  If  a 
septicaemia  supervenes  the  poisoning  becomes  extreme  and  the  organ- 
isms are  distributed  throughout  the  body. 

Immunity  following  recovery  from  natural  streptococcic  infection 


798   MICROBIOLOGY  OF  DISEASES   OF 'MAN  AND  DOMESTIC  ANIMALS 

is  very  slight  if  any,  and  never  of  a  permanent  sort.  Septicaemias 
once  established  are  generally  fatal,  and  erysipelas  can  recur  frequently. 

Bactericidal  substances,  opsonins,  agglutinins  and  precipitins 
have  been  demonstrated  in  immune  sera,  which,  however,  show 
little  therapeutic  success. 

Streptococci  are  eliminated  in  the  discharge  of  local  infections 
in  sputum,  etc.,  and  are  then  probably  more  virulent.  Infection  by 
contact  from  such  sources  is  particularly  dangerous.  In  anginas  and 
streptococcic  infections  of  the  respiratory  tract,  the  epidemiology  is 
practically  the  same  as  for  diphtheria  and  pneumonia.  Similarly 
erysipelas  is  to  be  treated  as  a  contagious  disease. 

In  the  prophylaxis  of  streptococcic  diseases,  greatest  care  must  be 
shown  where  chances  of  infection  by  the  virulent  strains  are  possible. 
Isolation  of  erysipelas  is  universally  practised  in  hospitals.  Similarly 
cases  of  puerperal  sepsis  and  any  local  disease  should  be  kept  from 
contact  with  other  puerperae.  Streptococcic  pus  from  all  sources 
is  to  be  carefully  destroyed. 

Streptococci  seem  to  be  always  present  on  the  exposed  surfaces 
of  the  body  and  are  probably  capable  of  giving  trouble  should  any 
local  lowered  resistance  occur.  The  prevention  of  this  may  be  accom- 
plished by  strict  antiseptic  treatment  of  wounds. 

PNEUMONIA* 
Streptococcus  pneumonia 

The  occurrence  of  a  diplococcus  in  the  large  majority  of  cases, 
especially  of  the  lobar  type  of  pneumonia,  has  caused  this  coccus  to  be 
regarded  as  practically  specific  and  warrants  the  name  of  Streptococcus 
pneumonia,  Diplococcus  pneumonia,  or  Pneumococcus.  As  occasional 
causes  of  pneumonia  should  be  mentioned  Streptococcus  pyogenes, 
Staphylo coccus  pyogenes  var.  aureus,  B.  coli,  Bact.  diphtheria,  Bact. 
influenza,  B.  capsulatus  mucosus  (pneumobacillus),  B.  typhosus  and 
Bact.  tuberculosis. 

Pneumonia,  is  world- wide  in  its  distribution  and  is  estimated  to 
form  anywhere  from  i  to  7  per  cent  of  all  cases  studied  in  internal 
medicine.  It  appears  to  be  more  frequent  in  regions  subjected  to 
sudden  changes  of  temperature  and  many  more  deaths  occur  in  the 
five  months  December  to  April  than  in  the  remainder  .of  the  year. 

*  Prepared  by  Edward  Pidlar. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      799 

The  incubation  period  is  two  or  three  days  of  rather  indefinite 
prodromata. 

The  onset  of  the  disease  is  marked  by  a  chill,  pain  in  the  side,  and 
rise  in  temperature.  The  respirations  become  frequent.  The  fever, 
as  a  rule,  runs  between  102°  and  io5°F.  for  from  five  to  ten  days  and 
then  in  favorable  cases  terminates  by  a  sudden  drop  of  temperature 
to  normal  within  a  few  hours  (crisis). 

The  most  striking  pathological  findings  are  a  marked  congestion 
and  oedema  of  the  lungs  following  which  the  lung  becomes  solid, 
airless  and  of  a  dark  red  color,  the  alveoli  showing,  microscopically, 
a  fibrinous  exudate  with  large  numbers  of  red  blood  cells,  some  leucocytes 
and  desquamated  epithelium.  Thereafter  the  lung  becomes  slightly 
softer  and  is  of  a  gray  color,  while  microscopically  the  red  cells  degener- 
ate and  leucocytes  are  much  more  in  evidence.  The  final  stage,  resolu- 
tion, is  marked  by  the  liquefaction  and  absorption  of  the  contents  of 
the  alveoli  and  the  entrance  of  air. 

Death  occurs  from  toxaemia  or  complications  such  as  carditis,  men- 
ingitis, etc.  Roughly  about  10  per  cent  of  all  deaths  are  due  to  pneu- 
monia and  the  fatalities  form  about  10  per  cent  of  the  total  number  of 
cases. 

The  Streptococcus  pneumonia  was  described,  as  found  in  the  spu- 
tum, by  C.  Frankel  in  1884. 

A  Gram-stained  preparation  of  the  sputum  is  sufficient  to  detect  the  diplococci 
but  cultures  are  necessary  for  positive  identification.  Some  medium  richer  than 
the  ordinary  by  the  addition  of  blood  or  serum  from  man  or  animals  is  best,  and 
may  be  inoculated  from  the  blood  and  organs  or  from  sputum  and  other  contami- 
nated sources  by  streaking  or  plating.  Injection  of  sputum  into  white  mice  or 
rabbits  will  often  cause  a  fatal  septicaemia  in  these  animals  and  the  coccus  may  then 
be  obtained  in  pure  culture  from  the  heart's  blood.  It  occurs  as  pairs  of  oval  or 
lanceolate  cocci,  with  their  contiguous  surfaces  somewhat  flattened  and  the  distal 
ends  slightly  pointed.  From  this  type  the  organism  may  vary  to  spherical  or  short 
bacillary  forms.  It  may  occur  also  singly  or  in  chains  of  varying  length  usually 
consisting  of  not  more  than  about  six  or  eight  individuals.  Well  developed  capsules 
which  may  surround  the  single  organism  or  the  pairs  and  chains  may  be  found  in 
exudates  or  in  milk  and  serum  media.  There  are  no  spores  nor  flagella.  The 
cocci  stain  readily  with  the  aniline  dyes  and  are  Gram-positive.  The  capsule  can 
be  demonstrated  by  several  methods  of  which  Welch's  and  Hiss'  are  the  most 
common.  The  temperature  range  is  from  25°  to  41°.  It  is  both  aerobic  and 
anaerobic,  and  grows  most  readily  in  a  medium  slightly  alkaline  to  phenolphthalein. 
Besides  serum  or  blood,  glycerin,  nutrose  and  dextrose  are  found  to  be  favorable 


800   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

for  its  growth.  On  agar  it  grows  in  small,  rather  transparent,  finely  granular 
colonies,  which  are  larger  and  more  opaque  when  serum  or  ascitic  fluid  is  present. 
Broth  is  faintly  and  uniformly  clouded.  Milk  is  a  favorable  medium  for  most 
strains  and  typically  is  acidified  and  coagulated.  On  potato,  growth  may  occur 
but  is  invisible.  Gelatin  can  rarely  be  used  at  a  temperature  high  enough  to  allow 
growth.  When,  occasionally,  growth  is  obtained  the  medium  is  not  liquefied.  On 
blood  serum,  growth  appears  as  small  clear  colonies  and  on  the  whole  is  better  than  on 
agar.  A  number  of  special  media  are  described  of  which  one  of  the  most  valuable  is 
the  inulin-serum- water  medium  of  Hiss.  It  typically  ferments,  with  the  production 
of  acid,  the  majority  of  carbohydrates,  even  polysaccharides  as  inulin.  On  blood 
agar  the  typical  organism  produces  a  greenish  zone  in  the  medium  about  the  growth, 
but  not  a  clear  zone  of  haemolysis  as  do  most  strains  of  streptococci.  The  dif- 
ferentiation from  other  streptococci  is  sometimes  a  matter  of  difficulty,  and  the 
following  characters  are  of  importance— the  lanceolate  shape,  capsule  formation, 
fermentation  of  inulin,  absence  of  haemolytic  powers,  agglutination  in  antipneu- 
mococcic  sera,  susceptibility  to  lysis  by  the  action  of  bile  salts.  Acid  is  an  im- 
portant and  characteristic  product  and,  if  allowed  to  accumulate,  rapidly  kills  the 
organism.  The  toxic  products  appear  to  be  closely  united  with  the  cell  bodies  and 
are  only  released  when  these  are  broken  up.  The  resistance  to  heat  is  not  great 
and  its  thermal  death-point  is  52°.  Light  is  germicidal  if  the  cocci  are  not  pro- 
tected in  thick  masses  of  sputum.  Drying  is  resisted  rather  well  in  sputum  or  the 
blood  of  infected  animals.  To  germicides  the  Pneumococcus  is  very  sensitive  and 
is  killed  in  a  few  minutes  by  the  common  disinfectants  in  their  usual  strength. 

The  pathogenic  properties  of  the  Pneumococcus  for  animals  is  some- 
what variable.  Natural  infection  is  not  common.  To  artificial  infec- 
tion mice  and  rabbits  have  been  found  most  susceptible,  while  guinea 
pigs,  dogs,  rats  and  cats  are  more  resistant,  and  birds  are  practically 
immune  probably  because  of  their  high  body  temperature.  Mice  are 
regularly  used  for  the  rapid  isolation  and  determination  of  pneumo- 
coccus  types.  By  special  methods  lobar  pneumonia  has  been  produced 
in  rabbits  as  has  also  endocarditis. 

Variations  in  virulence  of  the  Pneumococcus  are  very  marked.  The 
virulence  can  be  increased  by  passage  through  susceptible  animals. 
In  standardizing  type  sera,  strains  are  used  of  which  o.oooooi  c.c.  of  a 
broth  culture  will  kill  a  mouse. 

The  organism  gains  entrance  through  the  respiratory  mucosa  and 
as  a  matter  of  fact  appears  to  be  a  common  inhabitant  of  these  regions. 
However  the  organism  may  reach  the  lung  (the  lobar  distribution  sug- 
gests sowing  by  the  blood  stream),  it  is  certainly  frequent  to  find  posi- 
tive blood  cultures  during  the  disease — a  fact  which  accounts  for  the 
development  of  such  complications  as  meningitis,  endocarditis,  etc. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      8oi 

The  toxaemia  probably  arises  from  lysis  of  the  organisms  and  it  has  been 
shown  that  the  autolysis  of  cultures  in  salt  solution  gives  rise  to  a  soluble 
toxic  portion  and  an  insoluble  non-toxic  portion. 

Immunity  to  Pneumococcus  infections  can  be  shown  to  exist  after  an 
attack  but  only  for  a  short  time. 

Pneumococci  may  be  considered  as  inhabiting  the  mucous  mem- 
branes of  the  respiratory  tract  in  the  majority  of  people  and  acquire 
virulence  only  under  some  special  circumstances  lowering  the  general 
vitality.  In  pneumonia  and  some  kinds  of  bronchitis  as  above  men- 
tioned it  should  be  remembered  that  sputum  and  mouth  spray  may 
contain  large  numbers  of  virulent  organisms. 

Specific  therapeutic  agents  such  as  antipneumococcic  sera,  vac- 
cines of  dead  cultures  and  autolysates,  as  well  as  leucocy  tic  extracts,  have 
been  tried  and  all  with  some  promising  results.  The  earlier  failures 
with  serum  therapy  have  been  found  to  be  due  in  part  to  the  occurrence 
of  different  strains.  Four  strains  or  types  are  now  recognized.  About 
one-third  of  cases  is  due  to  Type  I,  one-third  to  Type  II,  10  to  15  per 
cent  to  Type  III  and  the  remainder  to  Type  IV.  Under  ordinary 
conditions  the  mortality  of  Type  I  and  II  is  25  to  30  per  cent,  of  Type 

III  59  percent,  and  Type  IV  10  to  15  per  cent.     The  corresponding 
antisera  are  more  successful  in  Type  I  infections. 

The  prophylaxis  of  Pneumococcus  infections  lies  in  general  hygienic 
measures,  in  the  destruction  of  sputa  and  avoidance  of  possible  infection 
by  mouth  spray,  etc.  In  households  in  which  pneumonias  existed  due 
to  Types  I  and  II,  Stillman  isolated  the  organisms  from  the  dust  iff 
43  per  cent  and  59  per  cent  respectively.  Occasional  carriers  of 
Types  I  and  II  are  found  not  associated  with  clinical  cases.  Type 

IV  predominates  in  the  mouths  of  the  healthy. 

ANTHRAX* 
Bacterium  anthracis 

Also  called  splenic  fever  or  charbon;  and  in  man,  wool-sorter's  dis- 
ease or  malignant  pustule. 

The  disease  has  been  known  for  centuries.  It  is  thought  that  it  was 
one  of  the  plagues  of  Egypt,  mentioned  as  a  murrain  on  beasts,  and  boils 
and  blains  on  man  and  beast.  The  first  accurate  characterization  of 

*  Prepared  by  P.  C.  Harrison. 
51 


8O2    MICROBIOLOGY   OF  DISEASES   OF   MAN   AND  DOMESTIC  ANIMALS 

the  disease  was  made  by  Chabert  about  1800.  Pollender  in  1849  and 
Rayer  and  Davaine  in  1850  reported  that  they  .had  seen  "filiform 
bodies"  in  the  blood  of  animals  which  had  died  of  anthrax,  and  in  1860 
Davaine  announced  he  had  succeeded  in  transmitting  the  disease  to 
healthy  animals  by  inoculating  them  with  blood  from  an  anthrax  in- 
fected animal,  and  asserted  that  these  filiform  bodies  or  bacteria  were 
the  cause  of  the  disease.  This  result  was  attacked,  and  for  ten  years 
there  was  a  fierce  controversy  over  this  idea,  which  was  finally  stilled 
by  the  convincing  experiments  of  Robert  Koch  in  1876.  Koch  culti- 
vated the  bacterium  of  anthrax  from  the  blood,  showed  that  the  inocu- 
lation of  these  cultures  in  susceptible  animals  produced  anthrax,  worked 
out  the  life  history  of  the  organism,  and  enunciated  the  cardinal  require- 
ments— which  constitute  the  proof  of  the  pathogenic  nature  of  an  organ- 
ism, what  later  bacteriologists  have  named  the  rules  or  postulates  of 
Koch. 

GEOGRAPHICAL  DISTRIBUTION. — The  disease  is  very  widespread, 
occurring  all  over  the  world  in  tropic,  semitropic,  and  temperate  cli- 
mates. Wherever  stock  are  found  in  large  numbers  anthrax  is  usually 
present.  The  disease  ravages  the  herds  and  flocks  in  Russia,  Siberia, 
India,  Argentina  and  parts  of  Hungary,  France  and  Germany.  Local 
epidemics  occur  constantly  in  England,  Canada  and  the  United  States. 
In  the  delta  of  certain  rivers  the  organism  probably  grows  in  the  soil 
as  in  the  deltas  of  the  Mississippi  and  Bramaputra,  and  the  disease  is 
also  common  along  the  banks  of  many  rivers  (Vistula,  Rhine,  Seine, 
etc.). 

The  anthrax  organism  is  a  large,  non-motile  rod,  from  5/x  to  io/i  long  and  IM  to 
i-5/i  broad.  In  cultures  it  frequently  forms  long  threads  or  filaments  (Fig.  167). 
The  free  ends  are  slightly  rounded,  but  those  in  contact  are  quite  square,  and 
slightly  larger  in  diameter  than  the  middle  of  the  cell.  Involution  forms  are 
obtained  by  culture  on  potato  or  at  temperatures  of  40°  to  42°.  It  forms  oval  spores 
without  distortion  of  the  mother  cell  (Fig.  168).  Free  oxygen  is  necessary  for  the 
development  of  these  bodies,  and  a  temperature  between  18°  and  41°.  Spore  ger- 
mination is  polar.  By  culture  at  42°  an  asporogenous  variety  is  formed.  It  stains 
readily  with  the  aniline  dyes  and  also  by  Gram's  method.  Under  certain  conditions 
a  capsule  may  be  seen.  The  organism  is  aerobic,  in  the  body  it  grows  as  a  faculta- 
tive anaerobe.  Its  optimum  temperature  is  37°,  minimum  12°,  maximum  45°. 
It  forms  characteristic  wavy  and  filamentous  colonies  on  gelatin  and  agar,  it  liquefies 
gelatin,  produces  an  arborescent  growth  in  gelatin  stab  cultures,  coagulates  and 
peptonizes  milk  with  an  alkaline  reaction.  Thermal  death-point  of  the  spores  in 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      803 

liquids  is  four  minutes  at  100°,  in  hot  air  140°  for  three  hours.  Mercuric  chloride, 
i  :  1000,  destroys  the  spores  in  a  few  minutes,  and  4  per  cent  carbolic  acid  with 
hydrochloric  acid  2  per  cent  in  one  hour. 

Zoologically,  anthrax  is  the  most  widespread  of  infectious  diseases; 
white  mice,  guinea-pigs,  rabbits,  sheep,  cattle,  horses  and  man  are 
susceptible.  Old  rats  are  insusceptible.  Von  Behring,  MetchnikofT 
and  others  have  shown  that  the  serum  of  white  rats  contains  a  lysin 


FIG.  167. — Bad.  anthracis.     Showing      FIG.  168. — Bad.  antkracis.     Spore  pro- 
the  thread  formation  of  colony.     (After  duction.     (After  Migula.) 

Kolle  and  Wassermann  from  Stitt.) 


capable  of  dissolving  the  bacterium  in  vitro.  Pigs  are  occasionally 
infected;  the  carnivora  generally  are  refractory,  the  bear  and  cat 
being  less  resistant.  Most  birds  are  insusceptible,  but  some  small 
birds,  like  the  sparrow,  are  more  susceptible.  Cold-blooded  animals 
are  refractory. 

Infection  occurs:  Through  the  food,  giving  rise  to  intestinal  anthrax. 
Cattle  and  sheep  are  usually  infected  in  this  manner  by  spores,  the  bac- 
terium being  destroyed  by  the  gastric  juice.  In  man  infection  through 
food  rarely  occurs. 

Through  the  air.  Infection  by  inhalation  through  the  lungs  occurs 
in  man  through  the  medium  of  dust  contaminated  by  anthrax  spores, 
hence  the  name  "wool-sorter's  disease." 

Through  wounds.  This  method  usually  occurs  in  man  and  also  in 
sheep.  Cutaneous  infection  comes  through  a  scratch  or  wound,  and 


804  MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

gives  rise  to  a  carbuncle — hence  the  name  "malignant  pustule."  It 
occurs  most  frequently  among  employees  of  tanneries,  wool-sorters, 
veterinary  surgeons,  and  those  whose  occupation  brings  them  into 
touch  with  infected  animals,  their  hides  or  products. 

The  incubation  period  is  a  short  one,  even  in  the  naturally  occurring 
disease;  inoculated  laboratory  animals  die  in  twenty-four  to  forty- 
eight  hours.  The  bacteria  appear  in  the  blood  about  fifteen  hours 
after  inoculation,  and  at  death  the  blood  simply  swarms  with  the  or- 
ganism. The  veins  are  turgid,  and  the  blood  is  often  very  dark,  and 


FIG.  169. —  Anthrax.     The  organisms  of  anthrax  in  the  capillaries  of  the  liver  of  a 
mouse.     {After  Williams.} 

coagulates  slowly.  The  bacteria  abound  in  the  capillaries  (Fig. 
169).  The  spleen  is  enlarged  and  contains  enormous  numbers  of  the 
organisms.  In  the  kidney  the  glomeruli  and  tubules  are  gorged 
with  the  bacteria,  which  pass  into  the  urine.  The  bacteria  can  pass 
into  the  milk  of  females  in  lactation.  The  bacteria  are  also  numerous 
in  the  liver,  lungs  and  mesentery,  but  few  are  found  in  the  muscles. 

Post-mortem  examination  of  subcutaneously  inoculated  laboratory 
animals  shows  subcutaneous  oedema  and  enlarged  spleen. 

The  organism  is  eliminated  from  the  body  in  urine,  faeces,  mucous 
discharges,  etc.  Pastures  become  infected  from  burying  anthrax 
carcasses  which  have  been  opened  or  have  been  skinned,  thus  favoring 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      805 

the  formation  of  spores.  If  buried  too  near  the  surface,  the  rise  of 
the  ground  water,  or  the  castings  of  earth  worms,  bring  spores  to 
the  surface  and  on  to  the  herbage,  where  they  may  be  ingested  by  graz- 
ing animals.  Tanneries  using  anthrax-infected  hides  may  be  the  cause 
of  distributing  the  organism  by  means  of  effluent  water  which  has  been 
used  for  steeping  hides.  Many  such  cases  have  been  traced  in  Dela- 
ware, Wisconsin  and  in  Ontario.  Hay  from  an  infected  pasture  may  be 
transported  to  a  distant  farm,  and  cause  an  outbreak  of  the  disease. 
In  Brazil,  vultures  feeding  on  anthrax  carcasses  disseminate  the  spores 
by  means  of  their  excrement,  and  thus  spread  the  disease.  Blood- 
sucking flies  may  also  be  instrumental  in  transferring  the  bacterium 
from  one  animal  to  another. 

.  Season  is  a  contributing  factor.  In  years  in  which  the  spring 
floods  have  been  very  high,  followed  by  a  hot  dry  season,  anthrax  is 
most  prevalent. 

There  are  a  few  preliminary  symptoms;  there  is  usually  sudden 
loss  of  appetite,  trembling  and  convulsive  movements.  Often  blood 
is  seen  in  urine  or  faeces  or  discharged  from  the  nose.  The  mucous 
membranes  are  often  bluish  in  color,  and  boils  or  pustules  may  occur 
on  various  parts  of  the  body.  Death  in  cattle  occurs  in  two  to  five 
days  and  in  sheep  in  twenty-four  to  thirty-six  hours.  The  mortality 
is  high  and  intestinal  cases  are  fatal  in  80  to  90  per  cent  of  the  animals 
attacked. 

The  usual  post-mortem  appearances  are  enlargement  of  the  spleen, 
blood  thick  and  tarry,  bloody  extravasations  in  the  muscles  and  organs, 
and  bloody  fluids  escaping  from  mouth,  nostrils  or  anus. 

In  anthrax-infected  districts  vaccination  should  be  used.  The 
vaccines  are  prepared  by  cultivating  the  bacterium  at  a  high  tempera- 
ture— 42°  to  43° — thus  forming  an  asporogenous  race,  according  to 
methods  devised  by  Pasteur  in  1881.  Two  vaccines  are  often 
used,  the  first  of  very  low  virulence,  the  second  more  virulent. 
Between  1882  and  1907,  8,000,000  sheep  and  1,300,000  cattle  have  been 
vaccinated  in  France  against  anthrax,  with  excellent  results.  Vaccina- 
tion by  toxin  has  been  advocated  by  Toussiant,  Hawkin,  Marmier 
and  others,  but  this  method  has  not  had  the  success  of  that  described 
above. 

For  treatment  of  the  disease  in  man,  Sclavo's  serum  has  been  of 
considerable  benefit.  This  serum  is  obtained  from  the  sheep  or  ass. 


806   MICROBIOLOGY   OF  DISEASES   OF   MAN   AND   DOMESTIC  ANIMALS 

The  animals  first  receive  the  two  vaccines  of  Pasteur,  then  more  viru- 
lent cultures  in  gradually  increasing  doses.  A  serum  is  then  obtained 
which  in  a  dose  of  2  c.c.  or  less  protects  a  rabbit  against  a  lethal  dose 
of  the  anthrax  organism. 

Animals  dead  of  anthrax  should  never  be  opened  or  skinned.  If 
doubt  exists  as  to  the  nature  of  the  disease,  an  ear  may  be  cut  off  and 
sent  to  a  laboratory  for  examination.  Anthrax-infected  carcasses 
may  be  either  burned  or  buried  at  a  depth  of  1.8  m.  (6  feet),  and  covered 
with  quick-lime,  and  as  an  extra  precaution  the  burial  ground  may 
be  fenced  off.  The  prime  necessity  is  to  prevent  the  formation  of 
spores,  as  it  has  been  shown  experimentally  that  they  remain  in  this 
condition  for  eighteen  years  and  produce  the  disease  when  inoculated. 
Soiled  litter,  forage  and  the  excretions  of  animals  dead  of  the  disease 
should  be  collected  and  burned. 

The  stalls,  stables,  implements  and  anything  that  has  been  in  con- 
tact with  the  diseased  animals  should  be  disinfected  by  burning,  boiling 
or-  the  use  of  some  disinfectant  like  5  per  cent  carbolic  acid. 

BACILLARY  WHITE  DIARRHOEA  OF  YOUNG  CHICKS* 
Bacterium  pullorum 

The  epidemic  type  of  diarrhoea  which  is  characterized  in  part  by  a 
whitish  diarrhceal  discharge,  and  which  is  now  known  as  "bacillary 
white  diarrhoea,"  is  caused  by  a  bacterium  which  belongs  to  the  colon- 
typhoid  group  of  organisms.  It  may  be  cultivated  easily  on  the  ordi- 
nary laboratory  media,  but  its  growth  on  slant  agar  containing  \yitte's 
peptone  is  delicate  and  bears  a  striking  resemblance  to  that  of  Strepto- 
coccus pyogenes .  This  finely  beaded  growth  is  an  important  aid  in 
the  identification  of  the  bacterium. 

The  specific  organism,  Bact.  pullorum,  is  present  in  the  liver,  lungs, 
kidneys,  spleen,  heart  and  unabsorbed  yolk  of  affected  chicks,  being 
most  easily  obtained  from  the  liver  and  yolk,  when  the  latter  is  present.. 
Some  of  the  most  common  post-mortem  appearances  of  the  organs  are 
those  of  the  liver  and  intestine,  the  former  showing  pale  and  congested 
areas,  while  the  intestine  is  colorless  and  to  a  large  extent  void  of 
contents. 

The  disease  seldom  manifests  itself  in  chicks  after  they  have  attained 
the  age  of  four  or  five  weeks.  The  greatest  mortality  usually  occurs 

*  Prepared  by  L.  F.  Rettger. 


MICROBIAL   DISEASES    OF   MAN  AND   DOMESTIC   ANIMALS      807 

within  the  first  two  weeks.  The  chicks  become  listless,  and  are  inclined 
to  huddle  together  for  warmth.  There  is  loss  of  appetite,  and  emacia- 
tion. The  wings  droop,  the  back  seems  to  shorten  and  the  abdomen 
protrudes  out  of  proportion,  causing  the  chicks  to  look  stilty.  The 
characteristic  whitish  discharge  from  the  bowel  may  be  absent  from 
individual  chicks,  but  is  usually  noticeable  in  groups  of  any  appreciable 
size. 

Bacillary  white  diarrhoea  may  be'transmitted  to  young  chicks  under 
five  days  old  through  infected  food  and  drinking  water,  as  has  been 
demonstrated  repeatedly.  Furthermore,  chicks  are  often  infected  with 
Bad.  pullorum  before  they  are  hatched.  This  is  due  to  the  fact  that 
the  yolk  of  infected  hens  carries  the  specific  organism  in  it  from  the  time 
of  its  formation  in  the  ovary.  Hence,  the  mother  hen  is  the  source  of 
infection,  having  retained  within  it  the  bacterium  in  question  from  the 
time  she  was  an  infected  chick,  or  having  acquired  it  later  in  life  through 
contact  with  diseased  fowls.  In  laying  hens  the  infection  is  localized 
in  the  ovary  which  becomes  decidedly  abnormal  in  appearance.  The 
partly  developed  ova  are  discolored,  misshapen  and  of  all  degrees  of 
consistency. 

Ovarian  infection  may  be  determined  by  the  macroscopic  agglutina- 
tion test  which  has  proven  itself  very  valuable  and  practicable  in  the 
organized  campaign  against  bacillary  white  diarrhoea  that  has  been 
conducted  in  the  State  of  Connecticut  for  the  past  six  years.  This 
method  of  diagnosis  has  been  found  to  be  much  more  valuable  than  the 
bacteriological  examination  of  eggs. 

Eradication  of  infected  laying  stock  is  the  solution  of  the  white 
diarrhoea  problem.  Flocks  which  are  at  all  doubtful,  or  which  have 
given  a  history  of  infection,  should  be  tested,  and  the  reacting  fowls 
eliminated.  Better  still,  no  eggs  should  be  used  for  hatching  which 
have  come  from  flocks  that  have  shown  an  appreciable  degree  of  infec- 
tion, although  reacting  individuals  have  been  removed. 

CHICKEN  CHOLERA* 
Bacterium  cholera  gallinarum 

The  bacterium  causing  this  disease  was  first  noticed  by  Perroncito 
and  Toussaint;  later,  in  1880,  it  was  described  by  Pasteur,  and  was  the 

*  Prepared  by  P.  C.  Harrison. 


808   MICROBIOLOGY  OF  DISEASES   OF  MAN   AND  DOMESTIC  ANIMALS 

first  organism  in  which  the  French  savant  succeeded  in  attenuating  the 
virulence  and  the  first  disease  for  which  a  vaccine  made  from  atten- 
uated organisms  was  prepared.  Koch  in  1878  described  an  organism 
of  similar  pathogenicity  as  the  bacterium  of  rabbit  septicaemia  and  in 
1886  the  term  hemorrhagic  septicaemia  was  given  by  Hueppe  to  a  num- 
ber of  infectious  diseases  of  the  lower  animals  in  which  hemorrhagic 
spots  were  found  in  the  tissues  and  internal  organs.  In  1900  Lignieres 
discussed  these  bacteria,  and  named  them  as  a  genus,  Pasteur  ellose,  the 
specific  name  given  depending  on  the  animal  for  which  it  was  most 
pathogenic.  Thus  he  distinguished  avian,  porcine,  ovine,  bovine, 
equine  and  canine  Pasteur elloses. 

The  specific  characters  of  this  group  are  small  ovoid  bacteria,  often  showing  bi- 
polar staining  when  treated  with  the  aniline  dyes,  non-motile,  no  spores,  Gram- 
negative,  polymorphic,  not  liquefying  gelatin,  no  visible  growth  on  naturally  acid 
potato,  milk  unchanged,  no  indol  production,  generally  aerobic  but  also  a  facultative 
anaerobe,  virulence  changeable,  but  usually  very  pronounced. 

The  bacterium  of  fowl  cholera, .Bact.  choleras,  gallinarum,  or  avian  Pasteurellose,  is 
from  o.s/t  to  1.25^1  long  and  0.25^  to  0.40/4  broad.  It  develops  best  at  37°,  and  very 
slowly  at  20°.  It  loses  its  virulence  hi  cultures  very  quickly,  and  it  succumbs 
readily  to  desiccation. 

The  disease  is  of  frequent  occurrence  in  Europe,  but  not  often  seen 
in  North  America  but  some  outbreaks  have  been  reported  in  the  United 
States  and  Canada.  Unfortunately  it  has  been  confused  by  poultry- 
men  with  any  disease  characterized  by  excessive  diarrhoea.  The  symp- 
toms first  noticed  are  the  yellow  color  of  droppings  soiling  the  cloacal 
feathers,  then  diarrhoea  sets  in,  the  character  of  the  discharge  varying, 
being  at  times  a  fluid  greenish  mass,  or  a  brown-red  mucus,  or  a  viscous 
transparent  and  frothy  fluid.  The  bird  becomes  uneasy,  drinks  copi- 
ously and  with  a  rise  in  temperature  to  42°  to  44°  the  bird  becomes 
drowsy  and  death  follows.  The  period  between  the  first  noticeable 
symptoms  and  death  varies  from  one  to  three  days.  Chronic  cases 
sometimes  occur  and  in  these  the  bacterium  is  found  with  difficulty. 
The  birds  become  infected  by  way  of  the  digestive  tract,  from  eating 
and  picking  up  material  infected  by  the  discharges  of  diseased  birds. 

Post-mortem  indications  are  blackened  combs,  congestion  of  the 
blood-vessels  in  the  organs  and  intestines,  and  punctiform  or  large 
hemorrhages  of  the  duodenum,  intestines  and  heart.  The  bacteria  are 
numerous  in  the  blood,  the  pulp  of  all  organs,  and  in  the  intestinal  con- 
tents. It  is  a  true  septicaemia. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      809 

If  the  disease  breaks  out  in  epidemic  form  the  best  and  quickest 
method  of  getting  rid  of  it  is  to  kill  off  all  the  fowls,  disinfect  the  houses, 
and  dig  or  plough  up  the  poultry  runs,  and  leave  them  two  weeks  before 
re-stocking. 

CHRONIC  BACTERIAL  ENTERITIS* 
Bacterium  panatuberculosis 

The  disease  produced  by  this  bacterium  has  been  demonstrated  in 
Germany,  Belgium,  Switzerland,  Holland,  Denmark,  and  perhaps 
other  European  countries.  It  is  known  by  various  names,  as  Johne's 
disease,  chronic  bacterial  dysentery,  and  chronic  bacterial  enteritis. 

This  bacterium  produces  a  chronic  infectious  disease  of  cattle  in- 
volving especially  the  intestinal  mucous  membrane  and  related  lymph 
glands.  Other  animals  do  not  seem  susceptible.  The  disease  produced 
is  usually  fatal.  Usually  the  most  conspicuous  general  symptom  is 
unthrift  in  spite  of  good  appetite  and  good  food  and  a  chronic  incur- 
able diarrhoea. 

This  microorganism  is  a  rod-shaped  bacterium  from  2/x  to  3^1  long 
and  about  0.5^  broad  and  is  strongly  acid-fast.  The  production  of 
active  toxins  is  to  be  presumed  since  the  amount  of  disturbance  is  fre- 
quently out  of  all  proportion  to  the  lesions  found  on  examination 
post-mortem.  The  period  of  incubation  has  not  been  defined,  but  is 
apparently  very  long. 

The  bacteria  are  present  in  the  faeces,  intestinal  mucosa,  and  sub- 
mucosa,  most  frequently  in  the  small  intestines.  The  large  intestines 
may  be  involved  later. 

This  microorganism  produces  chronic,  inflammatory  changes  of  the 
intestinal  mucous  membrane,  the  whole  intestinal  wall  becoming 
greatly  thickened. 

This  bacterium  resembles  closely  avian  tubercle  bacteria,  but  may 
be  distinguished  by  the  fact  that  the  avian  tubercle  bacterium  is  rather 
easily  grown  on  artificial  media.  This  organism  does  not  have  the 
same  pathogenic  peculiarities  as  the  avian  tubercle  bacterium.  It 
seems  well  demonstrated  that  many  cases  of  chronic  bacterial  enteritis 
do  probably  react  to  avian  tuberculin;  but  this  does  not  prove  identity. 

So  far  as  known  the  bacterium  is  eliminated  in  the  manure  of 

*  Prepared  by  M.  H.  Reynolds. 


8 10   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

affected  cattle  and  disseminated  in  this  way.     Wider  dissemination  is 
made  by  diseased  animals  moving  from  place  to  place. 

The  most  important  considerations  in  controlling  this  disease  are 
careful  disposition  of  contaminated  manure  and  isolation  of  suspected 
animals.  The  manure  should  be  used  only  where  it  can  not  serve  to 
spread  disease  to  other  cattle.  Sick  animals  should  be  carefully  isolated 
and  premises  thoroughly  disinfected. 

CONTAGIOUS  ABORTION  OF  DOMESTIC  ANIMALS* 
Bacterium  abortus 

The  premature  discharge  of  the  products  of  conception  from  the 
uterus  is  a  not  infrequent  occurrence  among  domestic  animals,  and 
doubtless  various  factors  may  from  time  to  time  operate  in  its  causation. 
Injury,  excessive  fermentable  food,  or  poisonous  food  may  at  times 
produce  this  result.  For  a  long  time,  however,  practical  husbandmen 
have  recognized  an  epizootic  form  or  a  contagious  abortion,  a  definite 
transmissible  disease,  of  which  the  loss  of  the  foetus  is  the  most  promi- 
nent characteristic.  This  disease  appears  to  be  generally  distributed 
in  all  agricultural  communities.  Cows,  especially,  are  affected,  but  a 
somewhat  similar  if  not  identical  disease  also  occurs  in  other  domestic 
animals. 

In  1897  Bernhard  Bang  discovered  in  the  uterine  exudate  of  a  cow, 
slaughtered  during  an  attack  of  this  disease  before  the  abortion  had 
occurred,  a  small  bacterium  which  he  was  able  to  grow  in  pure  culture, 
and,  by  inoculating  pure  cultures  of  this  organism,  he  produced  the 
disease  in  cows,  sheep,  goats  and  rabbits. 

The  microbe  is  a  short  non-motile  rod,  staining  with  moderate  ease, 
and  decolorized  by  Gram's  method.  It  does  not  form  spores  but  the 
vegetative  forms  are  fairly  resistant  to  drying  and  may,  perhaps,  live 
for  some  weeks  under  ordinary  conditions  in  pastures  and  stables.  Its 
artificial  culture  requires  special  technic  because  of  its  peculiar  oxygen 
requirement.  The  bacterium  usually  fails  to  grow  in  the  presence  of  the 
atmospheric  air  or  under  anaerobic  conditions.  It  requires  for  its  de- 
velopment a  partial  pressure  of  oxygen  somewhat  less  than  that  of  the 
atmosphere.  When  inoculated  into  deep  serum-gelatin-agar  tubes  and 
incubated  in  the  air,  the  colonies  develop  only  in  a  particular  zone 

•  Prepared  by  W.  J.  MacNeal. 


MICROBIAL  DISEASES   OF   MAN   AND   DOMESTIC   ANIMALS      8ll 

about  five  millimeters  beneath  the  surface  of  the  medium.  When  cul- 
tures are  placed  in  the  proper  atmosphere,  development  on  the  surface 
may  be  obtained.  Prolonged  cultivation  on  artificial  media  obscures 
this  peculiar  property  of  the  microbe  so  that  old  culture  strains  grow 
well  under  ordinary  aerobic  conditions. 

In  the  diseased  animal,  the  specific  bacteria  are  found  in  the  pla- 
centa and  amniotic  fluid,  frequently  also  inside  the  foetal  intestine, 
sometimes  in  the  tissues  of  the  fcetal  organs,  and  in  the  wall  of  the 
maternal  uterus.  The  placenta  appears  to  be  the  particular  organ 
favorable  to  the  development  of  the  germ,  and  when  this  has  been 
discharged  from  the  body  the  abortion  bacilli  no  longer  flourish,  al- 
though the  infection  may  continue  as  a  chronic  uterine  inflammation 
for  a  long  time.  The  general  health  is  only  slightly  disturbed.  At  the 
next  pregnancy  the  disease  is  practically  certain  to  reappear,  and  pos- 
sibly again  also  at  the  succeeding  one.  After  two  or  three  abortions  the 
animals  appear  to  have  acquired  an  immunity  to  the  infection,  and  may 
sometimes  breed  normally  thereafter,  although  some  animals  are  per- 
manently sterile  after  a  few  attacks  of  the  disease. 

The  organisms  escape  from  the  diseased  animal  in  the  products  of 
conception  at  the  time  of  the  abortion,  and  in  the  chronic  uterine  dis- 
charge which  may  continue  for  a  long  time  afterward.  The  disease  may 
be  conveyed  to  other  animals  by  contact  with  this  material  and  by 
eating  grass  or  other  feed  soiled  with  it.  Doubtless  the  male  is  an  im- 
portant factor,  possibly  the  most  important  factor,  in  transmitting  the 
disease,  although  no  serious  inflammation  is  produced  in  him. 

The  control  of  the  disease  depends  upon  the  isolation  of  the  infected 
animals,  cremation  of  infected  foetus,  placenta  and  discharges,  and 
thorough  disinfection  of  the  premises.  Heifers  and  healthy  cows 
should  not  be  allowed  to  mingle  with  cows  which  have  aborted,  nor 
should  they  be  served  by  a  bull  which  has  covered  infected  animals  at 
any  time.  Local  antiseptic  treatment  of  the  cow  which  has  aborted 
diminishes  the  danger  of  the  persisting  discharge. 

Contagious  abortion  also  occurs  in  other  domestic  animals,  espe- 
cially in  horses,  sheep,  goats  and  swine.  Inoculation  experiments  have 
shown  that  the  Bact.  abortus  of  Bang  can  infect  some  of  these  animals. 
Its  importance  as  a  factor  in  the  epizootics  of  abortion  occurring  natu- 
rally among  them  is  still  uncertain.  In  horses  at  any  rate  another  organ- 
ism appears  to  be  more  frequently  involved. 


8l2  MICROBIOLOGY  OF  DISEASES  OF  MAN  AND  DOMESTIC  ANIMALS 

DIPHTHERIA* 
Bacterium   diphtheria 

The  disease  is  epidemic  in  all  large  communities  especially  in  Europe 
and  America.  It  is,  however,  almost  absent  from  tropical  regions. 
Epidemics  and  pandemics  occur  in  cycles.  Essentially  diphtheria  is  a 
disease  peculiar  to  man.  Avian  diphtheria,  however,  is  known,  al- 
though seemingly  due  to  another  cause,  and  on  rare  occasions  natural 
infection  has  been  found  in  the  horse. 

The  period  of  incubation  is  said  to  be  two  to  five  days. 

In  man  the  disease  usually  begins  with  lassitude  and  fever  followed 
in  a  few  hours  by  "sore  throat."  The  inflamed  area  on  the  pharyngeal 
wall,  tonsils,  larynx  or  wherever  it  may  be  becomes  in  typical  cases  the 
seat  of  degenerative  changes  in  the  epithelium  and  underlying  tissues 
with  abundant  fibrinous  exudation  resulting  in  the  formation  of  a  com- 
paratively tough  membrane  or  pseudo-membrane,  which  is  a  striking 
and  characteristic  feature  of  the  disease.  This  local  lesion  is  almost 
always  found  on  mucous  membranes  though  occasional  instances  of 
infection  of  wounds  have  been  noted. 

In  connection  with  wound  infection  it  should  be  mentioned  that 
organisms  morphologically  resembling  the  true  B.  diphtheria  occur 
upon  the  body  surfaces  and  are  commonly  called  "diphtheroids." 
They  are  of  some  importance  because  occasionally  they  have  to  be 
distinguished  from  B.  diphtheria  by  culture  and  animal  inoculation, 
and  secondly,  while  of  doubtful  pathogenicity,  they  are  held  by  some 
to  be  responsible  for  a  certain  indolence  in  the  healing  of  wounds.  In 
an  investigation  by  Col.  Adami  and  others  in  the  Canadian  army  it 
was  found  that  in  some  localities  as  many  as  33  per  cent  of  war  wounds 
were  infected  with  diphtheroids  of  at  least  four  different  types,  while 
true  B.  diphtheria  occurred  in  less  than  0.6  per  cent. 

The  bacterium  of  diphtheria  was  described  in  1883  by  Klebs  in 
sections  of  typical  membranes.  The  organisms  were  isolated  and  dif- 
ferentiated in  1884  by  Loeffler,  who  was  able  to  fulfill  Koch's  postulates 
for  pathogenic  microbes.  Accidental  infection  of  the  human  being  has 
happened  in  the  laboratory  and  confirmed  the  findings  of  animal  inocu- 
lation. The  success  of  antitoxin  treatment  is  further  evidence  of  causal 
relationship. 

*  Prepared  by  Edward  Fidlar. 


MICROS IAL  DISEASES    OF  MAN  AND   DOMESTIC   ANIMALS      813 

The  organism  is  detected  in  the  following  manner:  A  sterile  swab  is  rubbed  gently 
over  the  inflamed  area  or  against  any  visible  membrane,  care  being  taken  to  touch 
other  parts  as  little  as  possible.  The  swab  is  then  immediately  inserted  into  a  tube  of 
specially  prepared  medium — Loeffler's  inspissated  blood  serum — over  the  surface  of 
which  it  is  rubbed  back  and  forth.  The  swab  is  returned  to  its  own  tube  or  left 
against  the  serum  and  the  culture  and  swab  sent  to  the  laboratory.  After  twelve  to 
eighteen  hours  of  incubation,  at  37°,  smears  are  made  from  the  cultures  and  stained 
with  Loefflers  methylene  blue.  The  diagnosis  is  made  on  the  morphological  char- 
acters of  the  bacterium.  Occurring  in  pure  cultures  the  form  of  the  bacterium  is 
subject  to  remarkable  changes  according  to  the  medium  and  length  of  cultiva- 
tion. Its  size  as  it  appears  in  exudates  and  from  serum  media  varies  from  i^  to 


FIG.  170. — Bacterium  of  diphtheria.     X  1000.     (After  Williams.) 


7/ji  in  length  and  0.25^  to  2/*  in  width.  The  rods  are  straight  or  slightly  curved, 
usually  not  uniformly  cylindrical  but  with  swellings  at  the  ends,  or  in  the  middle,  or 
irregularly  disposed  (Fig.  170).  Both  ends  may  be  rounded  or  both  pointed,  or  one 
rounded  and  the  other  pointed.  Branching  forms  are  not  infrequently  found. 
The  bacteria  may  appear  in  pairs  end  to  end;  more  frequently  and  typically  they  are 
inclined  to  one  another  at  a  greater  or  less  angle  and  may  assume  a  parallel  arrange- 
ment or  the  form  of  a  short  zigzag  chain.  The  arrangement  is  most  clearly  under- 
stood and  remembered  by  considering  the  peculiar  "snapping"  type  of  fission 
characteristic  of  the  group.  There  are  no  flagella  and  no  spores.  The  cell  mem- 
brane is  possessed  of  great  elasticity  as  evidenced  by  the  post-fission  movements. 
The  bacteria  stain  readily  with  all  the  aniline  dyes  and  retain  the  primary  stain  fairly 
well  by  Gram's  method.  With  Loeffler's  methylene  blue  they  stain  in  a  character- 
istically irregular  manner,  and  show  metachromatic  "granular"  forms,  "barred" 


814   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

and  "solid-stained"  forms  (Fig.  171).  On  a  basis  of  morphology  and  staining 
properties,  Wesbrook,  Wilson  and  McDaniel  have  devised  a  classification  which  is 
very  convenient  for  descriptive  purposes.  The  minimum  temperature  of  growth 
18°  to  19°,  optimum  35°  to  37°,  maximum  40°  to  41°.  The  bacterium  grows  most 
readily  in  the  presence  of  oxygen.  Under  certain  conditions  it  will  grow  anaero- 


Jv*  A 

,    l^y  vav, 

V-  ^ 


<f 

(<i 


FIG.  171. — Wesbrook's  types  of  Bad.  diphtheria,     a,  c,  d,  granular  types;  a',  c',  df, 
barred  types;  a2,  c2,  d2,  solid  types.     X  1500-     (From  McFarland.} 

bically.  The  optimum  reaction  of  blood  serum  media  is  about  +0.8.  The  amount 
of  acid  which  the  bacterium  can  endure  varies  with  the  kind  of  acid.  Gelatin  is  not 
liquefied,  neither  are  the  proteins  of  blood  serum  nor  of  milk.  Caseinogen  is  not 
changed  to  casein.  Some  carbohydrates  are  broken  up  with  the  production  of 
acid.  All  authorities  find  that  the  bacterium  forms  acid  from  dextrose.  It  is 
generally  agreed  that  acid  is  produced  from  lactose,  galactose  and  maltose.  Action 
on  dextrin,  lactose,  saccharose  and  glycerin  is  variable.  The  majority  of  workers  find 
mannit  is  unchanged.  An  acid  reaction  in  plain  broth  by  fermentation  of  muscle 
sugar  may  be  followed  by  the  production  of  alkali.  Gas  is  not  produced  under  any 
circumstances.  No  indol  is  formed.  Most  strains  cause  haemolysis  of  red  blood 
cells.  A  true  diffusible  toxin  is  formed  for  the  artificial  production  of  which  in 
broth  cultures  peptone,  absence  of  sugar,  an  alkaline  reaction  and  free  access  of 
oxygen  are  favorable  factors.  Growth  on  plain  nutrient  agar  is  not  so  abundant 
as  on  LoefHer's  blood  serum.  Colonies  of  two  types  may  be  found:  (a)  most 
common  is  small  grayish- white,  rounded,  slightly  raised,  almost  translucent  with 
more  or  less  granular  surface  and  dark  center,  the  margins  varying  in  irregularity, 
and  often  with  a  thin  extension  spreading  out  from  the  edge;  (b)  less  common, 
larger,  more  luxuriant,  white,  rounded,  raised,  granular  to  nearly  smooth  and  some- 
what moist.  Plain  broth  must  be  slightly  alkaline  to  litmus.  About  one-half  of 
cultures  grow  readily  and  half  very  feebly.  The  characteristic  growth  is  a  finely 
granular  deposit  at  bottom  and  along  sides  of  the  tube  leaving  the  broth  clear;  a 


MICROBIAL  DISEASES   OF  MAN  AND   DOMESTIC  ANIMALS      815 

few  cultures  produce  a  diffuse  cloudiness  with  more  or  less  well-developed  pellicle. 
Growth  on  gelatin  is  scanty  chiefly  owing  to  temperature  at  which  this  medium 
must  be  kept.  The  gelatin  is  not  liquefied.  In  milk  growth  takes  place  at  com- 
paratively low  temperature  (20°)  without  coagulation  but  with  acid  production. 
On  potato  growth  occurs  especially  if  slightly  alkaline;  in  the  majority  of  cases 
invisible;  it  may  appear  as  a  thin  dry  glaze  or  with  a  whitish  or  slightly  yellowish 
tinge.  On  Loeffler's  blood  serum  growth  is  rapid.  In  eighteen  to  twenty-four 
hours  colonies  are  rounded,  grayish-white  with  a  slightly  yellowish  tinge  moderately 
translucent  except  toward  the  center,  smooth,  moist  and  shiny.  The  margins  are 
only  slightly  irregular.  With  age  the  colonies  become  dull  and  opaque,  the  surface 
becoming  marked  by  concentric  lines  and  sometimes  also  exhibiting  radial  striation. 
Thermal  death-points  are  58°  to  60°  for  ten  minutes,  70°  for  five  minutes,  100° 
for  one  minute.  On  the  other  hand  —190°  for  seven  days  and  —252°  for  ten 
hours  have  failed  to  kill  in  some  instances.  Diffuse  light  hinders  growth.  Direct 
sunlight  kills  within  two  hours  to  a  few  days  according  to  the  medium  in  which  the 
organisms  are  suspended. 

The  organism  gains  entrance  into  the  body  through  the  mouth  and 
nose. 

The  bacteria  usually  remain  localized;  they  can  almost  always  be 
demonstrated  when  a  definite  membrane  is  present  and  often  when 
there  is  none.  They  are  practically  always  found  in  the  lung  of  fatal 
cases  because  of  direct  infection.  Entrance  of  the  bacteria  into  the 
blood  stream  with  resulting  infection  of  the  internal  organs  has  occurred 
in  fatal  cases. 

The  protective  apparatus  concerned  in  diphtheria  is  probably  dif- 
ferent at  the  beginning  from  that  involved  late  in  the  disease.  Experi- 
mentally, agglutinins,  bacteriolysins  and  opsonins  have  been  demon- 
strated in  exudates  and  serum.  While  these  properties  may  be 
important  in  warding  off  an  infection  they  appear  to  be  of  little  influ- 
ence once  the  bacteria  are  established,  and  thereafter  on  the  amount 
of  antitoxin  will  rest  the  outcome  of  the  disease. 

The  toxin  is  strongly  antigenic,  the  cell  bodies  feebly  so.  Aggressins 
have  not  been  demonstrated. 

The  organisms  escape  by  the  secretions  of  the  mouth  and  nose. 
Direct  infection  by  coughing,  sneezing  and  speaking  probably  takes 
place  frequently  not  only  from  the  sick  and  convalescents  but  also  from 
healthy  carriers. 

Control  of  the  disease  is  sought  by  quarantine  of  all  sick  persons 
and  the  placing  of  restrictions  if  not  actual  quarantine  on  those  exposed 
and  showing  the  bacteria  in  the  nose  and  throat. 


8l6   MICROBIOLOGY   OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

DYSENTERY* 
Bacterium  dysenteric^ 

Two  chief  types  of  dysentery  are  known,  one  due  to  a  protozoon, 
Entam&ba  histolytica;  the  other  due  to  a  bacterium — Bact.  dysenteries. 
Only  the  latter  is  here  dealt  with. 

Acute  dysentery  in  an  epidemic  form  is  found  chiefly  in  Asia,  some- 
times in  Europe  and  in  the  Philippines.  In  this  country  occasional 
small  epidemics  and  certain  types  of  summer  diarrhoeas  of  infants  have 
been  shown  to  be  due  to  Bact.  dysenteries.  The  disease  occurs  naturally 
only  in  man. 

Dysentery  is  an  intestinal  disorder  usually  acute  and,  in  its  epidemic 
form,  very  severe,  marked  by  a  flux  in  which  there  is  the  frequent  pass- 
age of  blood  and  mucus  with  severe  tenesmus  and  pain  in  the  abdomen. 
The  fever  accompanying  this  may  reach  104°  and  in  the  severe  cases 
delirium  and  death  may  result.  In  Japanese  epidemics  the  fatality  has 
reached  25  per  cent  or  more. 

The  pathological  findings  are  most  marked  in  the  intestine  where 
the  mucosa  is  swollen  and  hyperaemic,  with  more  or  less  haemorrhage 
and  extensive  necrosis. 

Shiga  in  1898  discovered  a  bacterium  in  the  stools  of  persons  suffer- 
ing from  the  disease  and  the  organism  was  agglutinated  by  the  blood 
serum  of  the  patients.  He  found  the  same  organism  repeatedly  in  a 
considerable  number  of  cases. 

The  results  of  many  other  investigations  have  demonstrated  the 
presence  of  several  forms  conforming  in  general  to  the  type  described 
by  Shiga  but  showing  some  difference  in  fermentation  properties;  these 
are  sometimes  known  as  para-  or  pseudo-dysentery  bacteria.  The 
most  important  of  these  are  the  Flexner,  Strong,  and  Hiss'  and  Russel's 
"Y"  types. 

The  constant  presence  of  the  organism  in  the  epidemic  type  and  the 
fact  of  agglutination  leave  little  doubt  as  to  the  etiological  relation.  A 
criminal  fed  with  a  pure  culture  of  Shiga's  organism  developed  the 
typical  disease. 

The  organism  can  occasionally  be  isolated  in  almost  pure  culture 
from  bits  of  mucus  in  the  stool.  Endows  and  other  special  media  may 

•  Prepared  by  Edward  Fidlar. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      817 

be  used  for  isolation,  but  ordinary  litmus  lactose  agar  plates  are  satis- 
factory for  Shiga  and  "Y"  types. 

Bad.  dysenteries  (Shiga)  is  rather  short  with  rounded  ends  and  closely 
resembles  the  typhoid  bacillus  morphologically  except  that  it  does  not 
possess  flagella. 


It  stains  readily  with  the  aniline  dyes  and  is  Gram-negative.  It  grows  best  at  body 
temperature,  is  aerobic  and  facultatively  anaerobic.  It  prefers  a  slightly  alkaline 
medium.  On  agar,  broth,  and  gelatin  growth  resembles  that  of  the  typhoid  bacillus. 
In  litmus  milk  an  alkaline  reaction  usually  follows  a  slight  primary  acidity  without 
any  further  apparent  change.  On  potato  growth  it  is  at  first  invisible  but  may  appear 
later  of  a  brownish  color.  Acid  is  formed  from  dextrose,  levulose,  and  galactose. 
(Other  types  described  differ  from  this  in  the  fermentation  of  mannit  and  sometimes 
of  maltose.)  Gas  is  never  formed.  Indol  is  not  formed.  (Other  types  usually  form 
indol.)  The  toxins  are  probably  chiefly  endo toxins,  though  soluble  poisons  have  also 
been  demonstrated  by  some  workers.  The  bacterium  remains  alive  for  months  when 
preserved  under  the  proper  conditions.  The  thermal  death  point  is  60°  and  re- 
sistance to  low  temperature  is  considerable.  It  is  sensitive  to  the  usual  strength  of 
ordinary  disinfectants. 


Dysentery  does  not  occur  in  animals  under  natural  conditions.  By 
artificial  methods,  however,  it  is  claimed  the  disease  has  been  repro- 
duced in  dogs.  Cultures,  living  or  dead,  are  often  extremely  toxic  to 
small  animals,  especially  the  rabbit,  and  produce,  after  intravenous 
injection,  violent  intestinal  symptoms,  due  evidently  to  the  excretion 
of  an  irritating  poison.  Nervous  symptoms  are  also  more  or  less 
marked  and  paralysis  sometimes  occurs  before  death.  Immunity  pro- 
duced artificially  in  animals  is  accompanied  by  the  production  of  lysins 
and  agglutinins  and  lately  antitoxins  have  been  described  in  accord 
with  the  demonstration  of  diffusible  toxins.  The  agglutination  in  man 
is  of  diagnostic  value. 

The  epidemiology  of  dysentery  is  the  same  as  for  typhoid  fever.  In 
a  few  instances  the  bacilli  have  been  demonstrated  in  the  faeces  of 
healthy  persons,  and  convalescents  may  remain  carriers  for  several 
months. 

Some  success  has  been  recorded  from  the  administration  of  animal 
immune  sera  and  has  been  attributed  to  both  lytic  and  antitoxic 
action.  Active  immunization  as  a  means  of  prophylaxis  does  not  seem 
to  be  of  much  value. 

52 


8l8   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

FOWL  DIPHTHERIA* 

This  disease,  popularly  known  as  Roup,  and  in  its  later  stages 
canker,  is  characterized  by  a  grayish-yellow  fibrinous  exudate,  called  a 
false  membrane,  which  forms  upon  the  mucous  membrane  of  the  eyes, 
nasal  passage,  mouth,  pharynx  and  larynx. 

Roup,  or  fowl  diphtheria,  may  be  caused  by  a  number  of  different 
organisms,  among  them  the  well-known  Ps.  pyocyanea  (green  or  blue 
pus  organism),  B.  cacosmus  and  other  species  which  give  rise  to  a  com- 
plex suppurative  process.  The  pus  formed  is  semi- solid,  cheese-like 
and  yellowish-white  in  color  without  any  tendency  to  become  soft  and 
liquid  or  to  perforate  the  surrounding  skin.  The  organisms  have  a 
tendency  to  penetrate  the  deeper  layers  of  the  mucous  membrane  or 
sub-mucous  tissues,  and  hence  swabs  or  cultures  taken  from  the  false 
membranes  may  not  contain  the  causal  microorganisms  which  are 
retained  in  the  depths  of  the  tissues.  Sections  of  membranes  from 
affected  fowls  show  large  numbers  of  pus  cells,  some  regular  masses, 
debris  of  epithelial  cells  and  bacteria,  and  thus  they  differ  from  the 
diphtheritic  membranes  of  man. 

The  organisms  mentioned  above  have  been  isolated  from  affected 
fowls,  not  only  in  America  but  also  by  Hauser  in  Europe.  Several 
investigators  have  described  other  bacteria  producing  false  membranes, 
and  there  are  a  few  who  think  that  coccidia  are  associated  with  the 
disease. 

Both  Ps.  pyocyanea  and  B.  cacosmus  are  able  to  produce  false  mem- 
branes when  inoculated  into  healthy  birds,  typical  croupous  and  diph- 
theritic membranes  in  the  mouth  and  eyes;  tumors  in  the  subcutaneous 
tissues,  the  contents  of  which  are  firm,  cheesy  and  yellowish- white; 
purulent  conjunctivitis,  blindness,  purulent  ophthalmia,  and  cheese-like 
exudations  in  the  bronchial  tubes.  These  indications  are  identical  with 
the  symptoms  of  "roup." 

The  disease  is  of  variable  virulence,  and  is  apt  to  become  chronic, 
especially  in  unhygienic  surroundings,  and  in  draughty,  badly  ventilated 
damp  houses.  A  common  cold  is  a  predisposing  factor,  and  favors 
the  invasion  of  the  organisms  mentioned. 

Treatment  of  severe  cases  is  useless,  and  demands  too  much  time. 
Diseased  birds  should  be  isolated  and  the  buildings  thoroughly  disin- 

*  Prepared  by  F.  C.  Harrison. 


MICROBIAL  DISEASES   OF  MAN  AND   DOMESTIC  ANIMALS      8lQ 

fected.  Slight  cases  may  be  cured  by  a  2  per  cent  solution  of  potassium 
permanganate,  in  which  the  bird's  head  is  plunged  for  a  few  seconds. 
This  treatment  should  be  given  twice  a  day  and  continued  until  all 
symptoms  have  disappeared.  The  most  effective  preventive  is  to  keep 
fowls  in  good  sanitary  conditions — in  dry,  clean  and  well-ventilated 
houses,  free  from  draughts. 

Besides  the  organisms  mentioned,  Loeffler  has  described  the  B. 
diphtheria  columbarium,  and  Loir  and  Duclaux  the  B.  diphtheria  gallin- 
arum  as  causing  fowl  diphtheria,  but  the  diseases  produced  by  these 
organisms  are  very  dissimilar  from  the  well-known  "Roup"  of  North 
America.  The  Klebs-Loeffler  bacterium  of  human  diphtheria  has  no 
pathogenic  effect  on  fowls. 

GLANDERS* 
Bacterium  mallei 

Glanders  is  a  very  common  and  serious  disease,  most  common 
among  equines.  It  is  communicable  to  the  human  being  by  inoculation 
and  by  the  same  process  may  affect  sheep,  goats,  and  laboratory 
animals.  Cattle  are  not  susceptible. 

Bact.  mallei  and  the  disease  it  produces  are  widely  scattered  over 
the  civilized  world  wherever  horses  are  numerous. 

This  infection  produces  a  disease  which  may  be  acute  or  chronic 
according  to  the  virulence  of  the  microorganisms  and  resistance  of  the 
animal.  Mules  and  donkeys  are  less  resistant  than  horses  and  usually 
have  the  disease  in  more  acute  form. 

The  characteristic  features  of  the  disease  produced  are  inflammatory 
changes  of  the  lymph  glands  and  lymph  vessels,  ulceration  of  mucous 
membranes,  the  tubercle,  the  farcy  bud,  the  lymph  cord,  and  the 
peculiar,  clear,  viscid  discharge.  There  is  considerable  fever  in  acute 
cases,  much  less  marked  or  absent  in  chronic  cases.  In  a  very  common 
type  of  the  disease  there  frequently  occurs  a  destructive  inflammation 
of  the  nasal  mucous  membrane  which  results  in  ulcers  and  consequent 
nasal  discharge. 

Glanders  in  man  is  rare  considering  the  frequent  opportunity  for 
infection.  There  are  usually  inflammatory  swellings  with  involvement 

*  Prepared  by  M.  H.  Reynolds. 


820   MICROBIOLOGY   OF  DISEASES   OF  MAN  AND   DOMESTIC  ANIMALS 

of  local  lymph  glands,  and  constitutional  disturbances  soon  follow  the 
local  symptoms.  Human  glanders  is  to  be  always  regarded  as  very 
serious  with  a  probability  of  fatal  termination.  Ulcers  may  develop 
in  the  nose  or  mouth  with  more  or  less  discharge.  Pustules  appear 
involving  the  skin,  and  abscesses  involve  deeper  structures  in  various 
portions  of  the  body. 

The  distribution  of  Bad.  mallei  in  the  animal  body  is  shown  by  the 
most  common  appearance  of  its  disease  in  the  skin,  subcutaneous 
tissue,  mucous  membranes,  lymphatic  system,  lungs,  liver,  spleen  and 
kidneys. 

The  etiological  factor  is  a  small  bacillus  with  rounded  ends  known 
as  Bact.  mallei,  discovered  by  Loeffler  and  Schiitz  and  several  others 
in  1882,  and  well  demonstrated  to  be  the  specific  cause  of  glanders. 

Entrance  is  usually  effected  by  way  of  a  mucous  membrane,  fre- 
quently the  intestinal,  sometimes  by  inoculation.  The  period  of 
incubation  seems  variable  and  uncertain  under  natural  infection,  but 
in  artificial  inoculation  with  virulent  cultures,  is  very  brief. 

Bact.  mallei  produces  toxins  in  artificial  media  and  also  in  body 
tissues;  the  well-known  preparation  called  mallein  may  be  considered 
in  this  class  awaiting  more  definite  knowledge.  This  substance 
produces  a  distinct  reaction  by  inoculation  into  glandered  animals, 
but  is  practically  non-toxic  for  healthy  equines.  So  far  as  known 
Bact.  mallei  attacks  the  animal  tissues  as  do  many  other  micro  organisms, 
the  harm  resulting  chiefly  from  bacterial  toxins  which  give  the  local 
tissue  reactions  leading  to  the  lesions  characteristic  of  glanders. 

In  its  action  on  tissues  Bact.  mallei  resembles  Bact.  tuberculosis; 
but  shows  a  more  rapid  development  of  lesions  and  more  active  in- 
flammation. 

Lesions  are  of  two  types — a  well-defined  nodule  followed  by  ulcera- 
tion  and  areas  of  diffuse  infiltration. 

The  nodule  as  it  appears  in  glanders  consists  largely  of  lymphoid 
cells  and  connective  tissue,  the  latter  increasing  as  the  case  becomes 
chronic.  Nodules  die  at  the  center,  suppurate,  and  discharge.  This 
occurs  especially  in  the  external  form  of  glanders,  which  affects  more 
commonly  the  legs  and  head.  Occasionally  defined  enlargements 
appear  in  the  involved  lung  areas.  Pulmonary  lymph  glands  are 
frequently  enlarged,  and  hardened.  The  superficial  skin  lesions  are 
in  the  form  of  nodules  previously  mentioned,  which  usually  suppurate 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      821 

and  ultimately  heal.  In  the  deeper  subcutaneous  tissues  there  is  a 
tendency  to  abscess  formation.  Small  nodules  or  tubercles  commonly 
appear  in  the  lungs  of  affected  horses.  These  vary  in  size  from  millet 
seed  to  as  large  as  garden  peas.  Various  degrees  of  broncho-pneumonia 
appear  and  more  or  less  pleurisy. 

Bad.  mallei  shows  no  flagella  and  is  non-motile.  It  is  a  small  bacterium  0.2 5/11  to 
o.4/i  thick  by  1.5^  to  3/x  long  with  rounded  ends  (Fig.  172).  Spores  have  not  been 
demonstrated.  It  is  generally  single.  Coccus  forms  sometimes  appear  and  even 
short  threads  when  grown  on  certain  media;  e.g.,  potato.  It  decolorizes  by  Gram's 
method  and  is  not  easily  stained  by  aqueous,  alkaline  aniline  dyes.  This  bacterium 
grows  fairly  well  between  25°  and  42°  on  potato,  glycerin  agar,  or  blood  serum.  The 


FIG.  172. — Bacterium    mallei.     From    pure    culture    on    glycerin    agar.     X  1000. 

(From  Migula.} 

guinea  pig  gives  a  reliable  diagnosis  by  inoculation,  showing  a  diagnostic  reaction 
within  four  or  five  days.  Diagnosis  may  also  be  confirmed  by  the  agglutination  test 
in  dilution  of  about  i  :  800  or  more  and  by  the  complement  fixation  test.  Satis- 
factorily stained  in  tissue  section  by  Kuehn's  carbol-methylene  blue.  Its  growth  is 
limited  at  an  upper  range  of  about  42°.  Bad.  mallei  is  difficult  to  isolate  by  culture 
methods  being  a  slow  grower  and  easily  lost  beside  faster  growing  organisms.  It 
can  be  better  isolated  by  guniea-pig  inoculation.  In  growth  it  is  both  aerobic  and 
anaerobic,  but  better  under  aerobic  conditions. 

The  virus  escapes  from  the  body  in  various  ways.  Elimination 
is  most  common  in  morbid  discharges  from  the  nose,  pharynx,  trachea , 
and  in  pus  from  farcy  buds  and  abscesses. 

Bad.  mallei  may  be  spread  directly  from  the  diseased  animal  to  the 
susceptible  animal,  or  the  dissemination  may  be  by  way  of  intermedi- 
ate objects;  e.g.,  troughs,  feed  boxes,  water  pails,  etc.  It  is  easily  killed 


822    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND   DOMESTIC  ANIMALS 

by  drying,  sunlight,  disinfectants,  and  heat;  but  may  remain  alive 
two  years  or  more  in  water  under  favorable  conditions.  Heating  to 
55°  kills  in  about  ten  minutes. 

In  man,  infection  occurs  usually  by  inoculation.  Cases  produced  in 
this  way,  occasionally  appear  among  laboratory  workers. 

All  plain  cases  of  glanders  in  domestic  animals  should  be  promptly 
destroyed.  Exposed  horses  should  be  tested  with  mallein.  Those  that 
react  should  be  destroyed  or  quarantined,  and  contaminated  premises 
properly  disinfected.  Immunization  has  not  been  satisfactorily  estab- 
lished. Diagnosis  of  doubtful  cases  is  made  by  the  use  of  mallein. 

INFLUENZA* 

Taking  influenza  with  its  complications,  the  recent  pandemic  in  its 
dimensions  and  the  swiftness  of  its  movements  has  been  one  of  the 
most  remarkable  diseases  in  the  annals  of  medicine.  Reliable  and 
complete  statistics  are  not  yet  available,  but  undoubtedly  the  world 
as  a  whole  will  have  to  record  deaths  by  the  millions  and  cases  by  the 
hundreds  of  millions,  and  these  chiefly  within  the  year  1918.  Its  wide- 
spread occurrence,  the  mystery  of  its  cause,  its  startling  infectiousness, 
and  its  later  high  mortality  formed  a  combination  which  in  psycho- 
logical effect  gained  it  a  place  nearly  comparable  with  an  old  time 
pestilence. 

It  has  seemed  at  times  as  though  the  public  felt  a  grievance  against 
the  medical  profession  for  its  comparative  ignorance  of  the  disease. 
Several  difficulties,  however,  have  attended  its  investigation.  Local 
outbreaks  so  suddenly  appeared  and  so  rapidly  declined  that  there  was 
insufficient  preparation  for  their  study,  or  where  preparation  was 
adequate  the  cases  would  disappear  before  a  definite  line  of  research 
could  be  foHowed  to  conclusion.  It  was  unfortunate,  too,  that  with  the 
doubtful  exception  of  the  monkey,  no  experimental  animals  were 
available  to  carry  on  the  work.  Finally,  it  has  been  difficult  to  de- 
termine what  constitutes  pure  influenza,  and  whether,  in  the  very 
fatal  pneumonias  which  latterly  appeared  with  it,  the  associated  organ- 
isms played  a  major  or  a  minor  part. 

Evidence  is  accumulating  that  the  bacterium  of  Pfeiffer  described 
below  and  still  known  as  B.  influenza  is  not  the  cause  of  the  disease,  but 
takes  the  role  of  a  secondary  invader  similar  to  that  of  the  pneumococcus 

*  Prepared  by  Edward  Fidlar. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      823 


and  streptococcus.  There  is  evidence  supporting  the  theory  of  a  fil- 
trable  virus;  a  bacillus  of  the  haemorrhagic  septicaemia  group  has  been 
described  as  a  probable  cause;  various  cocci  and  a  symbiotic  combina- 
tion of  organisms  have  also  been  suggested.  At  the  present  writing 
the  specific  infective  agent  must  be  regarded  as  unknown. 

Some  question  the  identity  of  the  recent  influenza  with  the  former 
disease  of  the  same  name  epidemic  in  1889  to  1892,  but  the  term  has 
been  rather  loosely  used  in  the  interval,  and  it  seems  probable  that  we 
have  had  a  recurrence  of  the  original  disease  but  in  more  virulent  form. 

Spain  is  given  by  some  as  the  place  of  origin  of  this  last  outbreak 
(whence  the  term  ''Spanish  Flu"),  and,  if  true,  it  seems  worthy  of  note 
that,  after  a  quiescence  of  twenty  years,  the  epidemic  revived  in  a  neutral 
country  rather  than  in  a  war-swept  country  where  some  sections  of  the 
population  were  living  under  abnormal  conditions  of  nutrition  and 
sanitation.  Once  started,  however,  war  conditions  certainly  hastened 
its  spread,  testified  by  the  explosiveness  of  some  of  the  outbreaks  in 
the  armies  where  the  intimate  contact  of  large  groups  of  men  was  so 
much  greater  than  in  civil  life.  The  swift  passage  from  person  to 
person  thus  afforded  to  the  specific  virus  and  its  concomitant  organisms 
may  to  some  extent  explain  the  remarkable  virulence  finally  attained. 

The  natural  disease  probably  occurs  only  in  man.  Reports  of 
epidemics  among  animals  have  not  yet  had  scientific  confirmation,  and 
the  experimental  disease  hi  the  monkey  needs  further  study. 

The  incubation  period  varies  from  about  one  to  seven  days. 

The  milder  type  of  influenza  which  occurred  in  the  spring  of  1918 
both  in  Europe  and  America,  seems  more  likely  to  have  been  the  pure 
infection  than  the  more  fatal  kind  appearing  in  the  following  autumn 
and  winter.  The  initial  stages  were  practically  the  same  in  the  two 
types,  but  in  the  winter  type  signs  of  lung  involvement  developed  with 
greater  or  less  rapidity. 

The  onset  is  sudden,  with  headache,  pains  hi  the  back  and  limbs, 
chilliness,  dry  throat,  suffusion  of  the  face  and  conjunctivas,  and  occa- 
sionally nose-bleed.  The  temperature  will  range  from  100°  to  io4°F. 
within  a  few  hours  but  the  pulse  remains  relatively  slow;  in  many 
groups  of  cases  the  blood-pressure  has  been  uniformly  low.  The 
leucocyte  count  is  below  or  at  normal  with  a  relative  increase  of  lym- 
phocytes. This  may  give  place  later  to  a  total  and  neutrophilic  in- 
crease as  pneumonia  develops.  Cough  may  be  absent  at  onset  but 


824  MICROBIOLOGY  OF  DISEASES  OF  MAN  AND  DOMESTIC  ANIMALS 

usually  develops,  dry  at  first  and  later  productive  as  pneumonia  sets 
in.  Herpes  sometimes  occurs.  The  prostration  and  depression  of 
influenza  are  a  characteristic  feature.  After  three  to  five  days  in  the 
milder  types,  the  temperature  comes  to  normal  by  lysis,  but  convales- 
cence as  a  rule  is  slow.  Cases  developing  pneumonia  are  usually 
marked  by  cyanosis,  a  secondary  rise  in  temperature,  a  change  in  the 
sputum  from  a  scanty  mucoid  to  a  purulent  and  often  blood-stained 
character,  an  increase  in  the  respiratory  rate,  and  the  appearance  of 
varying  physical  signs  in  the  chest. 

The  most  noticeable  pathological  findings  in  fatal  cases  occur  in 
the  chest.  Generally  speaking,  there  is  a  very  moist,  confluent,  lobular 
pneumonia  showing  haemorrhagic  zones  together  with  firmer  yellow- 
ish areas  large  or  small,  sometimes  lobar  in  extent  and  occasionally 
showing  fibrin.  The  cut  bronchioles  yield  a  thick  yellowish  pus  and 
often  appear  as  centers  of  greater  or  less  necrosis.  This  lesion  suggests 
that  of  the  purulent  bronchitis  described  by  English  writers  in  1916 
and  1917  but  of  more  advanced  character  and  with  an  added  pneu- 
monia. A  tracheo-bronchitis  is  present  which  is  usually  haemorrhagic. 
Haemorrhage  in  the  abdominal  recti  is  found  in  some  of  the  severe 
cases. 

There  is  no  doubt  that  the  specific  organism  enters  and  leaves  the 
body  by  the  mouth  and  nose. 

It  seems  probable  that  an  immunity  following  the  disease  may 
endure  for  at  least  a  few  months,  but  it  is  difficult  to  secure  data  for 
longer  periods. 

No  specific  substances  are  known  for  the  treatment  and  prevention 
of  the  disease  except  that  prophylactic  vaccination  against  the  second- 
ary organisms  such  as  B.  influenza,  the  pneumococcus  and  streptococ- 
cus, etc.,  have  been  tried  with  reported  success  both  in  England  and 
America.  The  mask  has  probably  been  an  aid  against  the  spread. 

Bact.  influenza 

This  organism  was  described  by  Pfeiffer  in  1892  as  occurring  in  large  numbers 
in  the  purulent  bronchial  secretion  expectorated  by  influenza  patients,  and  until 
the  present  pandemic  was  regarded  by  many  as  the  established  cause  of  the  disease. 
In  pure  cultures  the  bacterium  is  0.2^1  wide  by  o.5/x  long  with  occasional  threads  up 
to  2/j.  in  length.  Larger  forms  are  seen  on  boiled  blood  agar.  The  arrangement  is 
usually  single,  occasionally  in  pairs  end  to  end  arid  rarely  in  chains.  The  bacterium 
is  non-motile  and  does  not  show  spores  or  capsules.  It  does  not  stain  very  readily, 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      825 

sometimes  shows  polar  staining,  and  is  Gram-negative.  The  temperature  range  is 
about  26°  to  41  °C.  It  is  aerobic.  Haemoglobin  is  usually  regarded  as  an  essential 
constituent  of  media  for  its  growth.  Superheated  or  boiled  blood  agar  affords  more 
luxuriant  growth  than  agar  containing  unaltered  blood.  Colonies  are  small,  round, 
and  transparent  and  remain  discrete  unless  thickly  sown.  Growth  occurs  in  blood 
broth  used  in  thin  layers.  Resistance  is  less  than  the  majority  of  non-spore-bearing 
bacteria.  It  seems  especially  sensitive  to  drying  and  its  thermal  death  point  is 
60°  C.  for  about  one  minute.  The  bodies  of  the  bacteria  are  distinctly  pyogenic.  In- 
oculated animals  develop  agglutinins  and  complement-fixing  antibodies  which 
are  useful  for  identification  purposes.  Some  influenza  patients  give  positive  com- 
plement fixation  tests  with  antigens  of  B.  influenza,  but  the  same  may  be  said  of 
antigens  of  streptococci,  and  pneumococci.  There  are  probably  different  strains  of 
B.  influenza  as  in  the  case  of  pneumococci. 

WHOOPING  COUGH* 
Bacterium    pertussis 

According  to  latest  statistics,  the  death-rate  of  whooping  cough  is 
roughly  about  5.5  per  100,000  exposed.  The  causative  agent,  accord- 
ing to  Bordet  and  Gengou,  is  an  influenza-like  bacillus. 

It  is  a  non-motile  coccoid  bacillus,  stained  faintly  by  aniline  dyes 
and  Gram-negative.  It  is  distinguished  from  the  influenza  bacillus  by 
agglutination  and  complement  deviation  tests  and  by  the  fact  that  it 
can  be  gradually  adapted  to  ordinary  media. 

The  production  of  pertussis  in  young  animals  has  been  claimed. 
The  organism  has  an  endotoxin  which  produces  local  necrosis  after 
subcutaneous  injection. 

Further  evidence  on  the  etiology  of  whooping  cough  is  afforded 
by  the  observations  of  Mallory  and  others  who  have  found  large  num- 
bers of  small  microorganisms  corresponding  morphologically  with 
Bact.  pertussis  occurring  between  the  cilia  on  the  epithelial  cells  lining 
the  respiratory  tract  in  fatal  cases  of  the  disease. 

ILEMORRHAGIC  SEPTICAEMIA  f 

Bacterium  bomsepticum 

Haemorrhagic  septicaemia  belongs  to  a  class  of  similar  diseases 
grouped  under  the  general  head  of  Pasteur elloses. 

•  Prepared  by  Edward  Fidlar. 
t  Prepared  by  M.  H.  Reynolds. 


826   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

This  disease  has  been  reported  from  many  portions  of  North 
America,  from  some  sections  of  South  America  and  many  European 
countries.  It  is  known  under  a  variety  of  names,  as  cornstalk  disease, 
buffalo  disease,  pneumo-enteritis,  etc. 

Bad.  bomsepticum  produces  a  serious  disease  and  affects  a  wide 
variety  of  domestic  and  wild  animals.  The  domestic  animals  most 
commonly  affected  are  cattle,  sheep,  and  goats,  the  disease  being  much 
more  common  among  cattle  than  among  other  classes  of  stock. 

The  period  of  incubation  appears  to  be  short,  six  to  forty-eight 
hours.  The  onset  of  disease  is  usually  sudden,  and  the  case  acute. 
Haemorrhagic  septicaemia  does  not  spread  from  herd  to  herd  but  appears 
in  isolated  outbreaks  usually  at  wide  distances  apart.  It  is  a  common 
experience  to  find  a  serious  outbreak  in  one  herd  without  any  appear- 
ance of  the  disease  in  another  herd  in  an  adjoining  pasture,  with  only 
a  barbed  wire  fence  between.  Apparently  the  virus  exists  locally  and, 
under  as  yet  unknown  conditions  of  increased  virulence  or  lowered 
resistance,  is  able  to  start  a  local  outbreak.  Infection  is  often  im- 
ported with  infected  cattle  from  large  stockyards. 

Haemorrhages  found  at  autopsy  constitute  the  most  specific  and  char- 
acteristic clinical  evidence  of  this  disease.  Its  mortality  is  very  high, 
running  from  50  to  90  per  cent. 

Haemorrhagic  septicaemia  of  cattle,  chicken  cholera,  and  a  number  of 
other  diseases  belonging  to  this  group  are  very  similar  in  clinical  features 
and  the  bacteria  which  cause  these  diseases  are  very  similar  in  cultural 
and  microscopic  features.  Yet  all  evidence  points  to  the  fact  that  Bact. 
bomsepticum  acts  as  a  specific  causal  agent  for  haemorrhagic  septicaemia 
of  cattle. 

The  method  of  infection  is  still  uncertain,  probably  occurs  by  both 
inoculation  and  ingestion.  This  disease  does  not  appear  to  spread  easily 
by  simple  association  or  ordinary  contact  and  there  is  no  general 
atmospheric  distribution  of  Bact.  bomsepticum. 

Acute  and  rapidly  fatal  cases  where  the  autopsy  shows  only  trifling 
lesions  would  indicate  the  formation  of  active  toxins.  The  character- 
istic haemorrhages  indicate  the  production  of  substances  actively  toxic 
for  the  endothelial  cells  of  capillaries.  The  fact  that  these  haemorrhages 
vary  in  different  cases  from  extensive  subcutaneous  areas  to  those  that 
are  scarcely  visible  would  seem  to  indicate  that  this  toxin  is  produced 
in  greatly  varying  quantities  or  is  of  greatly  varying  toxicity. 


MICROS IAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      827 

The  lesions  produced  by  this  bacterium  indicate  a  general  distribu- 
tion through  the  body. 

The  characteristic  features  as  previously  mentioned  are  the  haemor- 
rhages which  are  either  subcutaneous,  submucous  or  subserous.  Lymph 
glands  are  frequently  infiltrated  with  extensive  haemorrhages. 

Cases  have  been  reported  as  showing  high  fever.  Those  studied  by 
the  writer  have,  as  a  rule,  showed  slight  disturbance  of  temperature 
until  near  death.  When  voluntary  muscles  are  involved  the  haemor- 
rhages invade  connective  tissues  rather  than  muscle  tissue  proper. 
Haemorrhages  of  the  pericardium  and  heart  wall  are  especially  common. 
The  rectal  and  vaginal  mucous  membranes  are  often  intensely  hyper- 
aemic  or  haemorrhagic.  Faeces,  urine  and  nasal  discharges  are  often 
blood  stained. 

Bad.  bovisepticum  resembles  so  closely  the  bacterium  of  chicken  cholera,  the  bac- 
terium of  rabbit  septicaemia,  Bact.  suisepticus  and  other  members  of  this  group  (Pas- 
teurelloses)  that  laboratory  differentiation  from  other  members  of  the  group  is 
exceedingly  difficult.  It  is  a  very  small  bacterium  with  rounded  ends,  closely 
resembling  a  diplococcus.  It  is  from  ifj.  to  I.$M  long  and  from  o.3/a  to  o.6/x  thick. 
Involution  forms  sometimes  appear.  It  shows  bipolar  stain,  decolorizes  by  Gram's 
method,  produces  no  spores,  has  no  flagella,  and  is  non-motile.  Short  chains  are 
not  uncommon. 

It  grows  best  at  body  temperature  and  slowly  at  room  temperature.  It  is 
killed  at  58°  in  eight  to  ten  minutes. 

The  disease  resembles  anthrax  in  some  general  characteristics  but 
is  easily  distinguished  by  microscopic  examination  of  the  blood  and 
failure  to  find  the  large  anthrax  bacterium  and  by  the  fact  that  the 
blood  from  the  general  circulation  is  apparently  normal  in  haemorrhagic 
septicaemia.  This  disease  also  resembles  symptomatic  anthrax  (black- 
leg) but  is  easily  distinguished  in  that  external  swellings  are  slight  if 
present  at  all  and  do  not  show  gas,  both  of  these  features  being  charac- 
teristic of  blackleg.  The  bacillus  of  symptomatic  anthrax  may  be 
recognized  by  microscopic  examination  as  so  different  from  Bacterium 
bovisepticum  that  there  could  be  no  mistaking  one  for  the  other. 

Little  is  known  concerning  elimination  of  this  bacterium  from  the 
diseased  body  and  concerning  methods  of  dissemination.  Hence  we 
are  very  much  in  the  dark  when  attempting  to  deal  with  the  disease 
produced  by  it. 


828   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

Isolation  and  disinfection  are  to  be  recommended  on  general  prin- 
ciples. Immunization  by  present  methods  appears  to  be  very 
questionable. 

LEPROSY* 
Bacterium  lepra 

Leprosy  is  a  disease  almost  as  old  as  history  itself  but  modern  leprosy 
cannot  be  definitely  identified  with  the  leprosy  of  the  Old  Testament, 
and  to-day  is  found  chiefly  in  oriental  countries  and  in  Norway,  Iceland 
and  Russia.  The  disease  is  present  in  some  of  the  provinces  of  Canada 
and  in  the  States  of  Louisiana,  California  and  Minnesota,  and  practic- 
ally limited  to  Scandinavians  in  the  latter  states.  The  natural  incuba- 
tion period  is  difficult  to  ascertain  but  is  probably  a  matter  of  months 
or  years. 

Clinically  there  are  two  main  types  of  the  disease,  the  tubercular 
or  nodular  and  the  anaesthetic  types.  In  the  first  form,  nodules  develop 
in  the  face  or  other  parts  of  the  body  usually  preceded  by  an  erythe- 
matous  patch.  The  mucous  membranes  become  affected  more  or  less 
extensively  and  the  hair  and  eyebrows  fall  out.  In  the  anaesthetic 
type  after  various  disturbances  of  sensation  which  may  sometimes  be 
followed  by  maculae  there  develop  areas  of  anaesthesia.  Bullae,  ulcers 
and  necrosis  may  occur  with  resulting  deformities  or  again  this  type 
may  exist  for  years  without  leading  to  such  results. 

The  bacteria  of  leprosy  were  first  described  by  Hansen  in  1879  and 
almost  at  the  same  time  Neisser  published  similar  descriptions.  Culti- 
vation of  Bact.  lepra  has  been  successful  in  the  hands  of  Clegg,  Duval 
and  others. 

The  microorganisms  can  be  shown  in  tissue  by  the  use  of  the  Ziehl- 
Nielsen  or  Gabbet  methods. 

In  tissue  the  bacterium  closely  resembles  the  bacterium  of  tuberculosis,  but 
usually  appears  somewhat  longer  (5^  to  7/1)  and  thicker  (about  O.SJLI)  straighter  and 
less  beaded.  Flagella  have  not  been  demonstrated.  The  bacterium  can  be  stained 
with  the  ordinary  aniline  dyes.  It  is  Gram-positive.  The  staining  reactions  on  the 
whole  are  like  those  of  Bact.  tuberculosis  but  Bact.  lepra  stains  more  readily  and  also 

*  Prepared  by  Edward  Fidlar. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS'    829 

decolorizes  more  readily;  30  per  cent  nitric  acid  followed  by  95  per  cent  alcohol  will 
totally  decolorize  them  while  Bad.  tuberculosis  resists.  1  he  optimum  temperature 
for  growth  ranges  from  32°  to  35°  when  grown  in  symbiosis  with  amoebae.  The 
reaction  of  the  media  upon  which  successful  isolation  takes  place  is  i  to  1.5  per  cent 
alkaline  to  phenolphthalein.  In  recently  isolated  cultures  growth  is  extremely 
slow  and  appears  on  the  surface  of  the  special  media  in  four  to  six  weeks  as  moist 
grayish-white  colonies  elevated  centrally,  with  an  irregular  wavy  margin  and 
attaining  a  diameter  of  2  mm.  Older  cultures  on  glycerin  agar  are  moist  and  abund- 
ant, and  develop  an  orange-yellow  pigment.  In  glycerin  broth  a  thin  membrane 
is  formed  at  the  surface  after  several  weeks,  while  a  small  amount  of  sediment 
collects  at  the  bottom  of  the  tube  leaving  the  medium  clear.  The  resistance  to 
heat  is  much  greater  than  that  of  ordinary  vegetative  bacteria,  so  that  cultures 
may  be  freed  from  contamination  by  the  latter  by  simply  heating  to  60°  for  one 
hour.  The  resistance  to  drying  is  probably  considerable. 


Human  leprosy  appears  to  be  confined  naturally  to  man  and  only 
lately  has  the  disease  been  transmitted  artificially  to  animals.  In  the 
Japanese  dancing  mouse,  and  less  frequently  in  the  white  mouse  and  the 
monkey  small  nodules  may  be  found  on  the  peritoneum  about  four  to 
eight  weeks  after  intraperitoneal  inoculation.  The  animals  do  not  show 
any  symptoms  of  illness  and  must  be  killed  in  order  to  find  the  lesion. 
More  recently  Duval  has  produced  an  apparently  typical  leprosy  in 
monkeys  by  repeated  injections  of  cultures  from  artificial  media. 

It  is  generally  considered  that  the  usual  path  of  entrance  of  the  bac- 
terium is  the  naso-pharyngeal  mucous  membrane.  The  organisms  seem 
to  be  distributed  slowly  over  the  body  and  according  to  their  location 
produce  the  different  types  of  the  disease.  They  are  found  in  the 
nodules  of  the  nodular  type  and  in  the  nerve  trunks  of  the  anaesthetic 
type. 

Agglutinins  have  been  demonstrated  in  the  blood  of  lepers.  Com- 
plement deviation  with  various  antigens  has  been  investigated  and 
indicates  an  antilipoid  immune  body  which  not  infrequently  gives  a 
positive  Wassermann  reaction.  Lepers  react  frequently  to  tuberculin 
inoculations  and  this  is  not  considered  to  be  always  due  to  associated 
tuberculosis. 

The  chief  source  of  elimination  of  leprosy  bacteria  is  the  nasal 
mucosa.  The  bacteria  have  been  demonstrated  in  this  region  in  about 
40  per  cent  of  the  macular  types,  80  per  cent  of  the  nodular  and  mixed 
types. 


830  MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

While  a  great  deal  of  popular  fear  exists  against  this  disease  it  is 
decidedly  less  infectious  than  pulmonary  tuberculosis.  Lepers  have 
unquestionably  been  subjected  to  a  great  deal  of  wholly  unnecessary 
persecution. 

Prophylactically,  isolation  has  certainly  demonstrated  its  value 
and  the  reported  increase  of  leprosy  in  certain  parts  of  Europe  has  been 
attributed  to  a  decrease  of  this  custom  of  segregation. 

PLAGUE* 
Bacterium  pestis 

Epidemics  have  been  recognized  since  the  second  century.  About 
half  the  population  of  the  Roman  Empire  died  in  the  sixth  century. 
An  epidemic  of  the  fourteenth  century  destroyed  half  the  inhabitants 
of  Europe.  In  India  during  1901  to  1904  about  2,000,000  died  of  the 
disease.  In  China,  in  Egypt,  South  Africa,  and  in  sea  ports  of  the 
Western  hemisphere,  plague  has  been  found. 

Among  animals  the  disease  has  been  found  chiefly  in  rats  and 
squirrels.  Dogs  may  occasionally  become  infected. 

Four  types  are  described,  the  ambulant,  bubonic,  septicaemic,  and 
pneumonic.  The  bubonic  type  forms  three-quarters  of  the  cases. 
Physical  and  mental  depression  accompanied  by  a  high  fever,  often  with 
a  remission  about  the  third  day,  occurs.  Collapse  may  then  follow  with 
death.  Glandular  swellings  (buboes)  appear  in  the  groin  and  axilla 
and  these  may  suppurate.  Haemorrhages  beneath  skin  and  mucous 
membranes  are  common.  The  third  type  is  a  very  rapid  form,  causing 
death  before  the  development  of  buboes.  The  fourth  type  is  also  a 
short  and  extremely  fatal  form,  marked  by  the  occurrence  of  broncho- 
pneumonia  due  to  the  plague  bacteria. 

The  bacterium  of  bubonic  plague  was  described  by  Yersin  and 
Kitasato  independently  in  1893.  They  found  it  in  glands  and  through- 
out the  body  in  fatal  cases. 

The  organism  is  readily  grown  from  the  buboes,  the  blood,  and  the  sputum  in  the 
pneumonic  type,  by  simple  inoculation  of  ordinary  media  of  a  slightly  alkaline  reac- 
tion. The  bacteria  are  i.5ju  to  i.yju  long  by  0.55/11  to  o.y/x  wide  with  rounded  ends 
occurring  singly  or  in  pairs  and  short  chains  in  exudates  and  sometimes  in  long 
chains  in  broth.  Involution  forms,  large  swollen  spheres,  clubs,  etc.,  are  char- 

*  Prepared  by  Edward  Fidlar. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      831 

acteristic  in  artificial  media.  There  are  no  spores.  It  is  non-motile.  Some 
observers  have  demonstrated  a  gelatinous  capsule.  Occasionally  very  distinct 
branching  occurs  (Hill).  It  stains  easily  with  aniline  dyes,  particularly  at  the 
poles  which  may  show  round  or  oval  granules.  It  is  Gram-negative.  Its  minimum 
temperature  for  growth  is  about  12°,  the  optimum  30°,  the  maximum  40°.  It  is 


FIG.  173—Bact.  pestis.     (After  Yersinfrom  Williams.) 


aerobic.  Agar  after  twenty-four  hours  shows  small  granular  grayish  colonies  with 
a  thickened  center  and  indented  margin.  Broth  shows  a  granular  deposit  and 
sometimes  a  pellicle  with  dependent  outgrowths,  the  medium  remaining  otherwise 
clear.  Gelatin  growths  are  as  on  agar,  and  the  medium  is  not  liquefied.  Litmus 
milk  may  show  slight  acid  formation  and  no  coagulation.  Potato  shows  nothing 
characteristic.  The  toxins  appear  to  be  largely  endotoxins,  though  soluble  poi- 
sons have  been  found  in  old  cultures.  No  indol  is  formed.  Resistance  toward 
heat  is  not  great,  boiling  kills  in  a  few  minutes.  Light  kills  in  a  few  hours.  They 
do  not  resist  drying  well,  but  in  a  moist  condition  remain  viable  for  over  a  year. 
The  usual  strengths  of  ordinary  disinfectants  kill  in  about  ten  minutes. 

Rats,  mice,  guinea-pigs,  rabbits,  and  monkeys  are  particularly 
susceptible  to  inoculation  and  even  insects  die  from  infection. 

The  bacterium  enters  the  body  through  the  (usually  abraded)  skin 
or  respiratory  tract.  After  involvement  of  the  nearest  lymphatic 
glands  the  bacteria  are  distributed  through  the  blood. 

Single  attacks  immunize.  The  antibodies  developed  are  agglutinins. 
probably  bacteriolysins,  and  possibly  antitoxins.  The  agglutination 
reaction  is  of  value  in  diagnosis. 


832    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

The  organisms  are  eliminated  in  the  exudates  from  suppurating 
buboes,  in  the  sputum  in  the  pneumonic  type,  and  are  present  through- 
out the  body  after  death.  The  dead  bodies  of  human  beings  and  of 
rats  are  sources  of  infection  for  other  rats.  There  seems  good  evidence 
of  these  animals  becoming  chronic  carriers  though  showing  no  symp- 
toms of  disease,  and  may  thus  be  important  factors  in  maintaining  and 
spreading  plague. 

The  disease  is  largely  communicated  by  means  of  fleas  which  have 
become  infected  by  living  on  other  human  beings  or  even  upon  rats. 

Prophylaxis  consists  of  isolation  of  pneumonic  cases,  thorough 
disinfection  involving  the  killing  of  fleas,  and  chiefly  the  destruction  of 
rats,  squirrels,  and  other  animals  which  may  serve  as  carriers.  Haff- 
kine's  vaccination  method  has  also  been  shown  to  be  a  valuable 
prophylactic  measure. 

The  serum  of  immunized  animals  has  been  tried  as  a  therapeutic 
agent  and  gives  encouraging  results  when  administered  in  the  early 
stages. 

SWINE  ERYSIPELAS* 
Bacterium  rhusiopathia  suis 

Swine  erysipelas  is  an  infectious  disease  of  hogs  characterized  by 
red  or  violet  discoloration  of  the  skin  and  mucous  membranes.  Swine 
erysipelas  does  not  exist  in  the  United  States  but  is  very  prevalent  in 
continental  Europe.  It  is  caused  by  a  very  small,  slender,  non-motile, 
non-spore-bearing  bacterium  (Bact.  rhusiopathia  suis)  which  stains  by 
Gram's  method,  and  grows  feebly  on  the  ordinary  culture  media. 
Development  is  best  under  anaerobic  conditions.  In  gelatin  stab 
cultures,  after  three  or  four  days,  a  white  growth  can  be  seen  along 
the  needle  puncture.  Radiating  from  this  are  a  number  of  delicate 
tufts  which  give  the  growth  the  appearance  of  a  fine  test-tube  brush. 
White  and  gray  mice,  white  rats,  and  pigeons  succumb  to  the  inocula- 
tion of  minute  amounts  of  the  culture.  The  bacteria  tend  to  collect 
within  the  bodies  of  the  leucocytes.  This  microorganism  is  closely 
related  to  and  possibly  identical  with  the  bacterium  of  mouse  septicaemia 
(Bact.  murisepticum) .  Preventive  inoculation  with  attenuated  cul- 
tures has  long  been  practiced  successfully  in  Europe. 

•  Prepared  by  M.  Dorset. 


MICROBIAL  DISEASES  OF  MAN  AND  DOMESTIC  ANIMALS     833 

TUBERCULOSIS* 
Bacterium  tuberculosis 

Consumption,  phthisis,  scrofula,  pearl  disease,  etc.,  are  synonyms 
of  the  term  tuberculosis. 

This  bacterium  in  its  several  varieties  produces  a  very  universal 
disease;  practically  all  common  animals  and  man  are  subject  to  it. 
Cattle  and  swine  among  the  domestic  animals  are  especially  susceptible 
to  this  infection  and  wild  animals  in  captivity  easily  become  affected. 

The  normal  progress  of  tuberculosis  is  slow.  Its  characteristic 
feature  is  the  tubercle  or  nodule  of  various  sizes. 

Tuberculosis  is  probably  the  most  common  and  serious  of  all  diseases 
for  either  animal  or  man. 

In  1918, 150,000  persons  died  from  tuberculosis  in  the  United  States, 
or  at  the  rate  of  150  per  100,000  population.  Based  upon  these  facts, 
it  is  estimated  that  about  10,000,000  of  those  now  living  in  the  United 
States  may  die  of  the  disease.  It  is  claimed  that  the  disease  alone  costs 
the  United  States  from  $400,000,000  to  $1,000,000,000  each  year 
(Fisher). 

If  the  loss  from  wage  earnings,  the  cost  of  the  patient  in  suffering, 
medical  treatment,  medicines,  nursing,  board,  and  care,  also  the  suffer- 
ing and  sacrifice  entailed  by  near  relatives,  friends,  and  communities 
are  considered,  the  loss  to  the  country  mentioned  above  does  not  appear 
so  enormous. 

It  is  estimated  by  the  United  States  Bureau  of  Animal  Industry  that 
2  per  cent  of  hogs  in  the  United  States  are  tubercular,  and  that  losses  of 
stock  in  the  United  States,  due  to  tuberculosis,  amount  to  $23,000,000 
annually.  Of  400,000  cattle  tested  in  many  states  of  the  United  States 
during  a  certain  period  9.25  per  cent  were  tubercular.  The  highest 
prevalence  of  tuberculosis  in  cattle  is  among  pure  bred  herds  and  in 
city  dairy  stables;  i.e.,  among  the  cattle  kept  most  closely  confined. 
It  is  most  common  in  old  cattle  and  rare  in  calves  under  six  months 
old.  Tuberculous  infection  is  quite  generally  scattered  among  cattle 
of  civilized  nations. 

Tuberculosis  appears  in  man  usually  in  the  form  of  lupus  (tubercu- 
losis of  the  skin),  or  scrofula  (tuberculosis  of  the  cervical  glands),  or 

*  Prepared  by  M.  H.  Reynolds. 
53 


834   MICROBIOLOGY   OF  DISEASES   OF   MAN   AND   DOMESTIC   ANIMALS 

phthisis  (tuberculosis  of  the  lungs).  It  also  frequently  appears  in  the 
mesenteric  glands  and  other  glands  of  the  body,  and  may  appear  in  any 
of  the  tissues.  It  is  quite  possible,  judging  from  autopsies,  that  many 
persons  have  tuberculosis  without  realizing  its  existence  in  the  body, 
and  without  its  being  detected  in  any  way.  It  is  questionable,  how- 
ever, whether  under  such  circumstances  the  disease  is  transmitted  or 
disseminated. 

As  a  rule  affected  cattle  show  no  definite  outward  signs  of  the  dis- 
ease. Badly  diseased  animals  sometimes  appear  poor  and  unthrifty. 
Many  cases  are  mild  and  latent.  A  few  tubercular  animals  cough; 
some  show  harsh  hair  and  skin  and  other  expressions  of  ill  health. 
While  these  symptoms  do  not  necessarily  indicate  tuberculosis,  they 
are  very  suggestive. 

Bact.  tuberculosis  may  invade  almost  any  tissue  or  organ  of  man  or 
the  animal  body  and  produce  a  variety  of  lesions.  Man  usually  gives 
some  evidence  of  the  disease  either  objectively  or  subjectively,  and  in 
many  instances  the  disease  assumes  a  definite  form  which  is  easily  rec- 
ognized by  medical  men,  unlike  its  presence  in  animals.  The  symp- 
toms are  more  evident  in  swine  than  in  cattle.  Affected  hogs  are  often 
unthrifty  and  show  glandular  enlargements  and  degenerations  of  the 
enlarged  glands. 

Avian  tubercle  bacteria  are  becoming  disseminated  among  poultry, 
and  to  a  serious  extent  in  some  sections  of  the  country.  Among  the 
more  prominent  symptoms  of  avian  tuberculosis  are  emaciation  with 
marked  anaemia  and  weakness.  Examination  of  the  carcass  shows  dis- 
ease most  frequently  in  the  liver,  but  intestines,  spleen,  lungs,  and  even 
the  skin  may  be  invaded.  Danish  authorities  report*  serious  outbreaks 
among  swine  due  to  avian  tubercle  bacteria. 

It  has  long  been  firmly  established  that  Bact.  tuberculosis  is  the  spe- 
cific cause  of  this  disease.  But  while  this  bacterium  is  to  be  regarded 
as  the  specific  cause  it  must  be  understood  that  this  organism  is  fre- 
quently associated  with  pus-producing  bacteria  which  are  responsible 
for  certain  phases  of  the  disease  as  commonly  seen.  It  should  be  under- 
stood also  that  persons  and  animals  become  more  susceptible  and  have 
greater  opportunities  for  infection  under  close  confinement  and  lack  of 
exercise.  There  has  been  great  difference  of  opinion  concerning  the 

*  Dunne,  Trans.  Jour.  Bd.  Agr.  (London),  22  (iQiSX  No.  I. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      835 

unity  of  the  tubercle  bacterium,  and  the  probability  of  inter-trans- 
mission between  man  and  the  lower  animals.  A  large  number  of  bac- 
teriologists now  hold  that  the  several  types  of  tubercle  bacteria  are  but 
environmental  variations  of  the  same  species.  In  any  case,  man  clearly 
appears  susceptible  to  both  human  and  bovine  types  at  least. 

The  entrance  of  the  germ  may  occur  in  four  ways,  namely,  by  way 
of  the  digestive  tract;  it  may  occur  by  way  of  the  respiratory  organs; 
it  may  occur  by  inoculation;  and  infection  may  possibly  occur  before 
birth.  Some  authorities  hold  that  the  most  common  infection  is  by 
way  of  the  digestive  tract  and  in  early  life.  Others  hold  that  inhalation 
tuberculosis  is  most  common. 

This  bacterium  produces  a  slow  toxaemia,  and  it  is  this  toxaemia  to- 
gether with  physical  embarrassment  of  the  vital  organs  by  extensive 
lesions  which  together  harm  the  affected  body.  Toxic  substances  are 
produced,  as  indicated  by  the  fact  that  killed  cultures  by  subcutaneous 
injection  may  destroy  local  tissues  and  produce  abscess,  debility,  and 
emaciation.  Production  of  toxins  is  indicated  by  the  further  fact  that 
certain  antitoxic  immunity  may  be  produced  by  minute  doses  of  killed 
culture  gradually  increased. 

Tuberculin  is  a  common  and  well  known  product  or  mixture  of  pro- 
ducts of  this  bacterium.  One  of  its  constituent  products  has  been  re- 
ported as  a  fever  producer.  Another  product  has  been  reported  which 
reduces  temperature,  and  still  another  which  produces  convulsions,  in 
sufficient  dose. 

Tuberculosis  may  be  very  general.  Almost  any  tissue  or  organ  in 
the  body  may  be  invaded;  but  as  a  rule,  not  many  organs  are  badly 
affected  in  the  same  case.  Distribution  occurs  by  way  of  both  the  blood 
and  lymph  streams,  especially  the  latter.  It  seems  probable  that  tuber- 
cle bacteria  may  be  distributed  in  the  body  by  wandering  phagocytes. 

The  Bact.  tuberculosis  has  a  characteristic  tendency  to  produce  tuber- 
cles or  nodules  which  may  be  large  or  small  and  which  have  a  tendency 
to  central  necrosis  and  degeneration.  Mucous  membranes,  under  this 
infection,  tend  to  develop  superficial  ulcers. 

The  lesions  produced  by  this  microorganism  may  vary  from  the 
tiniest  tubercles  to  extensive  areas  of  large  organs.  Lymph  glands 
frequently  enlarge  and  undergo  cheesy  or  calcareous  degeneration. 
Tubercular  masses  of  various  sizes  may  appear  upon  the  lining  mem- 


836   MICROBIOLOGY   OF  DISEASES    OF   MAN   AND   DOMESTIC   ANIMALS 

branes  of  the  chest  and  abdominal  cavities  and  upon  various  internal 
organs.  Cheesy  abscesses  may  appear  in  the  depths  of  soft  organs. 
In  cows  the  udder  is  occasionally  enlarged  and  shows  hard  masses 
with  little  or  none  of  the  heat  usually  occurring  in  connection  with  in- 
flammatory changes.  Bones  and  joints  are  often  involved  especially 
in  the  human;  these  structures  increase  in  size,  produce  pain,  and 
suppurate. 

Bacterium  tuberculosis  is  a  slender  rod-shaped  organism  with  rounded  ends  and 
under  certain  conditions  shows  granular  forms.  It  varies  between  2yu  and  SM  in 
length,  and  0.3/1  to  0.5^  in  width.  This  bacterium  is  usually  straight,  but  may  be 
bent;  it  appears  either  singly  or  in  groups  or  branched,  non-motile,  and  is  probably 
not  a  spore  producer  (Fig.  174).  Glycerin  agar,  blood  serum,  egg  slant,  and 
bouillon  may  all  serve  as  satisfactory  nutrient  media.  It  is  aerobic  and  its  tem- 
perature limits  for  growth  appear  to  be  29°-42°C.  With  the  exception  of  young 
and  rapidly  growing  forms  it  is  strongly  acid-fast.  Tubercle  bacteria  may  be 


'   l-> 


^ 

§P 

FIG.  174. — Bact.  tuberculosis.    Branching  FIG.  175. — Bacterium  tuberculosis. 

forms  from  a  culture.     (After  Migula.}        Sputum  preparation  uncolored.  (After 

Migula.} 

demonstrated  in  cover-glass  smears  from  diseased  tissues  and  fluids  and  in  tissue 
sections  (Fig.  175).  In  human  tuberculosis  the  bacteria  are  frequently  deter- 
mined in  the  sputum,  in  bovine  tuberculosis  the  bacteria  may  be  occasionally  dem- 
onstrated in  the  nasal  discharges  and  in  the  manure.  Positive  diagnosis  may  usually 
be  made  by  guinea-pig  inoculation.  For  microscopic  examination  a  cover-glass 
smear  is  fixed  in  the  usual  way,  then  stained  with  hot  carbol-fuchsin  three  to  five 
minutes  or  in  cold  stain  fifteen  to  twenty  minutes.  It  is  then  decolorized,  e.g., 
in  10  per  cent  nitric  acid,  and  counterstained  with  methylene  blue  for  about  one 
minute,  after  which  it  is  rinsed  and  ready  for  examination. 


MICROBIAL  DISEASES    OF   MAN   AND   DOMESTIC   ANIMALS      837 


It  is  conceded  that  tubercle  bacteria  do  not  multiply  in  nature  out- 
side the  animal  body  and,  therefore,  dissemination  must  depend  wholly 
upon  the  dissemination  of  infected  people  or 
animals  and  materials  infected  by  diseased 
men  and  animals.  Tubercle  bacteria  escape 
from  open  ulcers  or  from  tubercular  lesions 
which  connect  with  digestive  or  respiratory 
organs.  They  may  reach  the  surface  in  other 
ways;  e.g.,  by  the  discharge  of  abscesses. 

In  controlling  tuberculosis  among  humans 
at  the  present  time,  several  methods  are  in 
vogue.  In  some  localities,  an  effort  is  made 
to  segregate  tuberculous  patients  during  the 
day  for  the  purpose  of  treating  them  as  well 
as  teaching  them  how  to  care  for  themselves. 
This  method  aims  to  instruct  how  to  prevent 
dissemination  and  transmission  of  the  disease, 
to  prepare  suitable  nourishment,  and  to  secure 
the  advantages  of  open-air  influences.  This 
instruction  not  only  extends  to  the  patients 
but  others  with  whom  the  patients  may 
mingle.  Sanitaria  are  also  constructed  to 
receive  patients  suffering  from  the  disease, 
and  care  for  them  under  suitable  medical 
supervision  by  proper  treatment,  nourish- 
ment, and  open-air  life.  Again,  the  policy 
is  being  inaugurated  to  instruct  tuberculous 
patients,  where  it  is  impossible  to  reach 
them  by  other  means,  to  care  for  themselves 
in  their  own  homes. 

By  these  general  hygienic  measures,  much 
good  has  been  accomplished,  not  only  for  the 
patients  but,  also,  in  a  diminution  of  the 
number  of  new  cases  developing. 

The  animal  disease  is  carried  to  distant 
points,  most  commonly  by  breeding  stock. 

Locally   the   disease  spreads  either  by    the     ,  FIG  176.— Bad. 

.      „  losis.  Glycerin  agar  culture 

movement  of  affected  cattle,  or  frequently     (After  Curtis  from  Stilt.) 


838   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

by  infected  milk.  Hogs  receive  their  infection  from  the  milk  of 
tubercular  cattle  or  from  the  manure  or  carcasses  of  such  cattle  in 
feeding  yards.  Unventilated  stables  are  favorable  for  the  spread  of 
this  disease  because  with  insufficient  ventilation  the  bacteria  are  not 
carried  out,  but  become  constantly  more  numerous.  The  tubercle 
bacterium  is  quite  resistant  to  drying,  but  is  rather  sensitive  to  sun- 
light. It  is  usually  destroyed  by  moist  heat  in  six  hours  at  55°;  in 
twenty  minutes  at  60°;  and  generally  in  five  to  twenty  minutes  at 
95°,  depending  upon  the  protection  it  may  have. 

Conditions  of  sensible  sanitation  are  of  the  utmost  importance. 
These  include  exercise,  sunlight,  and  ventilation,  particularly  sunlight. 
In  order  that  effective  control  work  may  be  done  among  animals, 
tuberculin  must  be  used  freely  and  conscientiously. 

The  method  of  dealing  with  diseased  herds  depends  upon  breeding 
and  value.  Common  cattle  are  usually  dealt  with  most  economically 
and  efficiently  by  slaughter  with  a  view  to  using  such  carcasses  as  may 
pass  inspection.  Valuable  cattle,  especially  pure  bred  animals,  may  be 
used  for  breeding  purposes,  gradually  building  up  a  sound  herd  and 
gradually  displacing  the  diseased  animals.  This  latter  plan  is  usually 
unprofitable  and  unwise  except  for  very  valuable  cattle. 

FOOT  ROT  OF  SHEEP* 
Bacillus    necrophorus 

This  is  an  infectious  disease  of  sheep  characterized  by  an  ulcerative 
inflammation  of  the  tissues  just  above  the  horny  part  of  the  cleft  of  the 
hoof.  It  is  seen  in  Europe,  England,  Australia,  and  the  United  States. 
Sheep  are  made  lame  and  if  the  disease  is  not  checked  by  appropriate 
treatment,  the  hoof  becomes  greatly  distorted,  the  sheep  being  finally 
unable  to  walk.  Mohler  and  Washburnf  state  that  foot  rot  is  caused 
by  B.  necrophorus,  this  organism  being  associated  with  pus-producing 
micrococci.  B.  necrophorus,  which  is  a  strict  anaerobe,  tends  to  grow 
out  into  long  filaments;  it  is  stained  by  the  ordinary  aniline  dyes,  but  not 
by  Gram's  method.  Rabbits  and  white  mice  are  susceptible  to  inocu- 
lations of  this  bacillus,  but  guinea-pigs  appear  to  be  immune.  The 
disease  is  treated  by  causing  infected  sheep  to  walk  through  a 
disinfecting  solution,  such  as  a  3  per  cent  solution  of  carbolic  acid. 

*  Prepared  by  M.  Dorset. 

t  Bull.  63.  Bur.  An.  Industry,  U.  S.  Dept.  Agr.,  1904. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      839 

MALIGNANT  (EDEMA* 

• 
Bacillus  oedematis  maligni 

The  disease  occurs  as  the  result  of  infection  of  wounds  with  dust  or 
soil.  The  wounds  must  involve  the  tissues  deeply  as  in  compound 
fractures  and  deep  cuts. 

Any  animal  may  be  infected,  although  the  dog  and  cat  are  said  to  be 
rather  more  resistant  than  others.  The  guinea  pig  is  very  susceptible. 

The  incubation  period  is  short,  from  one  to  two  days  as  a  rule. 

The  usual  case  begins  with  sudden  spreading  haemorrhagic,  sub- 
cutaneous oedema  and  high  fever.  Practically  no  gas  is  formed.  The 
fluid  shows  bacilli  both  with  and  without  spores.  Where  soil  contami- 
nation exists,  mixed  infections  with  gangrene  are  common. 

Pasteur  in  1877  and  Koch  and  Gaffky  in  1881  found  and  studied 
the  organism  and  by  passing  from  animal  to  animal  established  the 
causal  relationship. 

Glucose  agar  or  glucose  gelatin  is  inoculated  with  the  suspected  fluid,  plates 
poured  and  placed  under  anaerobic  conditions.  The  organism  is  o.8/t  to  IM  wide. 
Filaments  may  occur.  The  rods  without  spores  are  uniform  in  width  with  slightly 
squared  ends.  They  are  usually  single,  though  pairs  end  to  end  are  frequent  and 
chains  are  also  found.  Oval  spores  are  formed  somewhat  variable  in  their  position, 
with  a  diameter  usually  larger  than  that  of  the  vegetative  rod,  bringing  about  a 
spindle  shape.  Peritrichic  flagella  have  been  demonstrated,  about  twenty  in 
number.  It  stains  readily  with  aniline  dyes,  usually  Gram-negative  though  some- 
what variable  and  indefinite  in  this  regard.  Growth  takes  place  at  both  20°  and 
37°.  It  is  a  strict  anaerobe.  Like  anaerobes  in  general  it  prefers  the  presence  of  a 
fermentable  carbohydrate  such  as  glucose.  On  agar  the  colonies  are  small,  whitish, 
and  irregular  in  outline.  Gelatin  and  blood  serum  are  digested,  caseinogen  is 
changed  to  casein  which  is  then  digested.  In  both  protein  and  carbohydrate  media 
a  gas  is  produced  which  has  a  very  disagreeable  odor.  The  spores  are  very  resistant. 

This  resistance  accounts  for  its  continuous  presence  in  earth  and 
dust  and  as  a  constant  inhabitant  of  the  intestine  of  animals,  especially 
of  herbivora. 

SYMPTOMATIC  ANTHRAX  OR  BLACKLEG! 
Bacillus  anthracis  symptomatici  (Bacillus  chauvcei) 
Blackleg,  black  quarter,  symptomatic  anthrax,  quarter  ill,  are  syno- 
nyms employed  to  designate  this  disease. 

*  Prepared  by  Edward  Fidlar. 
t  Prepared  by  M.  H.  Reynolds. 


840   MICROBIOLOGY   OF  DISEASES   OF   MAN   AND  DOMESTIC  ANIMALS 

Symptomatic  anthrax  is  a  very  old  disease  and  until  recent  years  has 
been  confused  with  true  anthrax.  This  disease  is  widely  distributed, 
affecting  practically  all  countries  and  climates. 

It  is  enzootic,  never  spreading  widely  or  rapidly,  and  is  often  found 
in  certain  infected  valleys  and  in  relatively  small  areas.  Young  cattle, 
generally  under  two  years  of  age,  are  most  commonly  affected,  but 
sheep  and  goats  are  susceptible  to  this  infection. 

This  disease  is  infectious  by  inoculation,  perhaps  also  by  in- 
gestion,  and  usually  acute.  Subcutaneous  and  muscular  tissues  are 
especially  affected.  Its  most  prominent  and  characteristic  feature  is 
swelling,  affecting  most  frequently,  the  front  or  hind  quarters,  and 
not  extending  below  the  knee  or  hock.  As  a  rule,  the  bacillus  of  symp- 
tomatic anthrax  produces  a  very  acute  disease  with  severe  constitu- 
tional disturbances,  and  early  death. 

The  bacillus  of  symptomatic  anthrax  has  been  clearly  demonstrated 
to  be  the  specific  cause  of  blackleg.  The  period  of  incubation  in  the 
natural  disease  is  uncertain.  Under  artificial  inoculation  this  period 
varies  from  two  to  three  days  and  is  occasionally  as  short  as  one  day. 

This  bacillus  produces  in  culture  a  very  active  toxin.  This  toxin 
is  quite  resistant  to  heat.  That  the  bacillus  of  symptomatic  anthrax 
stimulates  the  production  of  antibodies  and  that  the  injury  is  done  by 
toxins,  is  shown  by  the  fact  that  immunity  against  virulent  culture  may 
be  produced  by  treatment  with  presumably  sterile  filtrates  of  virulent 
cultures. 

The  bacillus  of  symptomatic  anthrax  is  rarely  found  in  the  general 
blood  before  death;  but  is  abundant  in  the  affected  muscle  and  over- 
lying subcutaneous  tissue.  It  also  occurs  in  great  numbers  in  the  bile 
and  intestinal  contents. 

Mucous  membranes  become  congested  and  then  very  dark.  There 
is  a  high  fever.  Local  swellings  occur  which  are  at  first  sensitive  and 
later  insensitive  and  gaseous.  There  is  usually  developed  a  very 
marked  swelling  of  a  front  or  hind  quarter  or  of  the  neck,  with  rapid 
formation  of  gas.  The  serous  membranes,  particularly  the  pleura  and 
peritoneum,  develop  severe  inflammation  with  haemorrhages  and 
infiltrations  and  corresponding  exudation  in  the  cavities.  General 
decomposition  is  rapid  and  the  swelling  may  show  a  slight  acetone 
odor.  The  local  subcutaneous  tissues  are  infiltrated,  haemorrhagic  or 
gaseous.  The  local  lymph  glands  are  swollen  and  haemorrhagic  or 


MICROBIAL  DISEASES   OF  MAN  AND   DOMESTIC  ANIMALS      841 

oedema tous.  Muscle  fibers  show  various  degenerative  changes. 
The  abundant  gases  are  mostly  hydrogen  and  carbon  dioxide.  Blood 
from  the  general  circulation  is  normal  as  to  color  and  coagulation. 

B.  anthracis  symptomatici  is  about  3/4  to  6/x  long  by  0.5/1  to  o.8/i  thick.  This  is 
a  spore-bearing  bacillus  of  drum-stick  shape  or  spindle  shape  and  is  anaerobic.  It 
grows  best  at  about  37°.  It  stains  either  by  the  simple  aniline  dyes  or  by  Gram's 
method.  In  artificial  cultures,  it  sometimes  shows  long  forms.  This  organism  is 
motile  for  a  short  time,  but  soon  loses  this  power,  probably  on  account  of  the  oxygen 
to  which  it  is  exposed.  It  shows  well-defined  flagella  and  develops  spores.  The 
specific  organism  may  be  demonstrated  by  the  microscope  in  the  blood  without 
staining  if  done  soon  after  death. 

The  bacillus  of  symptomatic  anthrax  is  easily  demonstrated  in 
cover-glass  smears  from  the  affected  tissues,  and  is  very  different  from 
the  bacteria  of  anthrax  and  haemorrhagic  septicaemia,  the  only  diseases 
liable  to  be  mistaken  for  blackleg  excepting  malignant  oedema. 
Anthrax  is  aerobic.  Symptomatic  anthrax  is  anaerobic.  This  organ- 
ism may  also  be  demonstrated  by  animal  inoculation.  The  guinea-pig 
serves  well  for  this  purpose;  it  is  very  susceptible  to  inoculation  and 
gives  a  characteristic  blackleg  reaction  in  both  symptoms  and  lesions. 
From  the  lesions  thus  produced  the  characteristic  bacilli  are  easily 
demonstrated  by  the  microscope. 

Elimination  of  this  virus  from  the  body  occurs  chiefly  in  the  various 
discharges,  and  especially  in  the  manure  and  also  in  general  decompose 
tion  of  the  carcass.  Dissemination  of  this  disease  is  chiefly  if  not  ex- 
clusively by  diseased  carcasses  and  parts  of  carcasses  and  by  the  dis- 
charges. Contaminated  soil  plays  a  very  important  part  in  the 
prevalence  of  this  disease.  It  appears  possible  that  the  specific 
bacillus  may  even  multiply  in  the  soil. 

Carcasses  should  be  burned  if  possible,  otherwise  very  deeply  buried 
and  covered  with  lime.  Contaminated  grounds,  or  stable  floors  must 
be  thoroughly  disinfected,  for  the  infection  is  very  persistent  and  difficult 
to  eradicate  except  by  most  vigorous  effort  since  the  spores  are  very 
resistant  to  heat  and  drying.  Preventive  inoculation  after  the  method 
of  Arloing  as  improved  by  Kitt  is  very  satisfactory.  Their  vaccine 
consists  of  specially  treated  muscular  tissues  from  the  diseased 
part. 


842   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

TETANUS* 
Bacillus  tetani 

This  disease  is  found  throughout  the  world  but  more  frequently  in 
warmer  than  in  colder  climates.  Certain  localities  are  particularly 
affected.  Man  and  domestic  animals  are  susceptible. 

The  incubation  period  varies:  a  few  hours  in  the  case  of  small  ani- 
mals Deceiving  injections  of  toxin;  several  days  or  weeks  in  cases  of 
natural  infection  in  man,  or  even  several  months  as  in  some  cases  of 
wounded  soldiers  who  had  received  injections  of  protective  serum. 
Tetanus  has  followed  operations  in  which  old  healed  wounds  have  been 
opened  up. 

Under  natural  conditions  the  disease  follows  a  wound  of  a  punc- 
tured type  with  contamination  by  earth,  especially  in  injuries  of  hands 
and  feet. 

It  is  characterized  by  tonic  spasms  of  the  voluntary  musculature 
usually  beginning  in  some  one  group  of  muscles  and  finally  becoming 
general.  The  parts  first  affected  are,  in  cases  artificially  produced, 
those  at  the  site  of  inoculation,  but  in  natural  infections  in  man  it  is 
more  common  for  the  disease  to  manifest  itself  by  stiffening  of  the 
muscles  of  the  neck  and  face,  producing  what  is  ordinarily  termed  "  lock- 
jaw." In  less  severe  infections  in  man  local  pain  and  stiffness  are  the 
first  indications.  The  spasms  occur  in  paroxysms  which  are  spon- 
taneous or  excited  by  effort.  They  are  more  or  less  prolonged  and  ex- 
hausting and  are  accompanied  by  greater  or  less  pain.  Death  results 
from  general  loss  of  strength  or  involvement  of  the  respiratory  muscles. 
The  shorter  the  incubation  period  the  higher  the  mortality.  Few 
recover  when  the  incubation  period  is  less  than  ten  days,  about  half  the 
cases  recover  when  the  period  is  more  than  fifteen  days.  In  the  British 
army  in  the  first  two  years  of  the  war,  the  mortality  ran  over  50  per 
cent,  and  after  that  about  25  per  cent. 

The  nerves  may  show  injury  as  indicated  by  swelling  and  redness 
and  microscopically  nerve  cells  have  been  observed  in  a  state  of  granu- 
lar degeneration;  there  is  a  more  or  less  distinct  general  congestion  of 
the  organs. 

While  lockjaw  has  been  known  clinically  for  centuries,  it  was  not 

•  Prepared  by  Edward  Fidlar. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      843 

until  1884  that  the  infectious  character  was  demonstrated  when  Carlo 
and  Rattone  and  Nicolaier  were  successful  in  animal  inoculations. 
Kitasato  obtained  pure  cultures  of  the  bacillus  in  1889. 

The  organisms  may  be  detected  rarely  by  examination  of 
stained  preparations  of  the  pus  from  the  wound.  Pure  cultures  may 
be  obtained  by  inoculating  an  alkaline  dextrose  broth  with  pus  or  tissue, 
incubating  under  anaerobic  conditions  for  about  forty-eight  hours  until 
sporulation,  then  exposing  half  an  hour  to  a  temperature  of  80°  to  kill 
all  vegetative  forms  and  subsequently  making  subcultures.  If  other 
spore-bearing  bacteria  are  present  considerable  difficulty  may  be  en- 
countered. Subcutaneous  inoculations  of  mice  or  guinea-pigs  is  a  good 
method  for  demonstrating  the  presence  of  the  organism,  but  pure 
cultures  should  be  combined  with  some  aerobe  (say  B.  coli)  to  secure 
results. 


FIG.  177. — Tetanus  bacilli  showing  end  spores.     (After  Kolle  and  Wassermann  from 

Still.) 

The  B.  tetani  is  about  2/z  to  5/i  in  length  by  0.3^1  to  o.8/n  in  width  with  rounded 
ends.  The  vegetative  rods  are  uniformly  cylindrical  but  the  terminal  spores  give  a 
"drum  stick"  appearance  (Fig.  177).  The  arrangement  is  usually  single,  but 
threads  may  occur  especially  in  old  cultures.  The  organism  forms  round  terminal 
spores  which  have  a  diameter  of  IM  to  1.5/1*.  The  young  bacilli  are  motile  and  possess 
50  to  70  peritrichic  flagella.  Motility  is  lost  with  sporulation.  The  bacillus  is 
stained  by  the  aniline  dyes  and  is  Gram-positive.  The  spores  are  readily  dem- 
onstrated by  the  special  stains.  The  range  of  temperature  for  growth  is  from  about 
14°  to  45°  with  an  optimum  about  37°.  The  organism  is  usually  considered  an 
obligate  anaerobe  though  experimentally  aerobic  strains  have  been  developed  but 
with  loss  of  pathogenic  and  toxogenic  properties.  Pure  cultures  do  not 


844   MICROBIOLOGY   OF   DISEASES    OF   MAN  AND   DOMESTIC   ANIMALS 

develop  in  an  atmosphere  of  carbon  dioxide.  Media  for  the  cultivation  of  the 
bacillus  should  be  slightly  alkaline  and  should  contain  for  best  growth  about  2 
per  cent  of  glucose  or  1.5  per  cent  sodium  formate.  The  addition  of  pieces  of  fresh 
raw  sterile  tissue  is  valuable.  On  agar  at  37°  colonies  appear  in  forty-eight  hours 
which  show  microscopically,  a  mass  of  tangled  threads  resembling  colonies  of  B. 
subtilis  or  Bact.  anthracis.  In  broth  a  cloudiness  is  produced  in  twenty-four  to 
thirty-six  hours  with  the  development  of  gas  and  a  very  disagreeable  odor.  In 
gelatin  the  colonies  develop  more  slowly  than  on  agar  and  show  liquefaction.  In 
old  stab  cultures  a  pine  tree  growth  occurs.  Gas  is  usually  produced.  In  milk 
growth  occurs  without  coagulation.  Acid  is  produced  in  some  carbohydrate  media. 
Gas  is  produced  during  the  action  upon  protein  and  consists  chiefly  of  carbon  dioxide 
but  also  of  hydrogen  sulphide  and  certain  volatile  organic  compounds  commonly 
found  in  putrefactions.  The  tetanus  bacillus  forms  two  soluble  toxins,  tetano- 
lysin,  and  tetano-spasmin.  The  former  is  less  stable  and  dissolves  red  blood- 
corpuscles.  The  latter  produces  the  characteristic  spasms  of  the  muscles.  This 
poison  may  be  obtained  after  one  to  two  weeks'  growth  in  slightly  alkaline  salt- 
peptone-bouillon  under  anaerobic  conditions  at  37.5°  and  separated  by  filtration 
through  porcelain  niters.  When  taken  by  the  mouth  the  toxin  is  ineffective,  given 
intravenously  it  produces  a  generalized  tetanus,  while  after  subcutaneous  injection 
the  disease  begins  with  local  spasms.  The  central  nervous  system  is  reached  by 
ascent  of  the  toxin  along  the  motor  nerves  nearest  the  point  of  inoculation.  A 
dose  of  toxin  injected  directly  into  -the  nerve  trunk  of  an  animal  may  produce  a 
fatal  result  when  it  is  innocuous  intravenously.  The  spores  often  withstand  80° 
for  one  hour  and  live  steam  for  about  ten  minutes.  Direct  sunlight  destroys  them 
in  time.  They  survive  drying  for  several  years  and  resist  the  ordinary  disinfectants 
for  a  considerable  length  of  time,  i :  1000  mercuric  chloride  for  three  hours,  5  per 
cent  carbolic  acid  for  about  ten  hours. 

Practically  all  mammalia  are  susceptible  to  tetanus  though  rats  are 
but  slightly  so.  Very  minute  doses  of  toxin  suffice  to  kill  mice  and 
guinea-pigs.  Birds  show  but  little  susceptibility  and  the  hen  is  said 
to  be  three  hundred  thousand  times  more  resistant  to  tetanus  toxin 
than  the  horse.  Reptiles  and  amphibians  are  practically  immune  to 
very  large  doses  when  kept  at  low  temperature. 

Natural  infections  probably  do  not  occur  without  the  presence  of 
other  microorganisms.  The  bacillus  and  its  associated  material  gains 
entrance  through  some  break  in  the  tissues.  The  organism  is  prac- 
tically confined  to  the  site  of  inoculation,  but  it  is  sometimes  found  in 
the  blood  and  internal  organs  after  death. 

Against  toxin-freed  cultures  phagocytosis  is  probably  the  process 
which  overcomes  infection.  The  toxin  is  highly  antigenic  and  animals 
can  be  immunized  against  it  in  a  manner  similar  to  that  for  diphtheria 
toxin. 


MICROBIAL  DISEASES    OF   MAN  AND   DOMESTIC   ANIMALS      845 

While  direct  infection  of  one  person  from  another  has  occurred, 
cases  of  human  tetanus  are  very  rarely  responsible  for  others. 

Horses  and  cattle  are  chiefly  responsible  for  its  distribution,  the 
tetanus  bacillus  being  common  in  manure,  which  accounts  for  the 
occurrence  of  tetanus  in  soil-contaminated  injuries. 

Tetanus  antitoxin,  as  a  prophylactic  measure,  is  widely  and  success- 
fully used  in  all  suspicious  wounds  in  civil  life,  and  was  extremely 
valuable  during  the  war.  Its  administration  should  be  combined  dur- 
ing the  first  twelve  hours  after  injury,  with  thorough  surgical  treatment 
aimed  at  the  removal  of  damaged  tissue  and  foreign  bodies.  The  pas- 
sive immunity  begins  to  decline  in  about  ten  days.  The  serum  is  less 
effective  as  a  therapeutic  measure,  and  must  be  given  as  soon  as  pos- 
sible after  the  detection  of  symptoms  in  very  large  doses,  intrathecally, 
intravenously  and  subcutaneously. 

TYPHOID  FEVER* 

Bacillus  typhosus 

Typhoid  fever  is  one  of  the  most  widespread  of  bacterial  diseases 
and  is  found  endemic  in  practically  all  the  countries  of  the  world. 
Epidemics  frequently  occur  because  of  the  infection  of  some  local 
public  utility  related  to  food  or  drink,  particularly  water  or  milk. 

Typhoid  fever  occurs  naturally  only  in  man.  Intraperitoneal  inocu- 
lation of  susceptible  animals  may  result  in  death  with  acute  peritonitis, 
but  lesions  are  in  no  way  specific  and  can  be  produced  by  the  colon 
bacillus. 

The  period  of  incubation  varies  ordinarily  from  five  to  twenty-one 
days,  with  an  average  of  fourteen  days. 

The  first  week  of  the  disease  in  man  begins  with  a  train  of  rather 
indefinite  symptoms  such  as  headache,  loss  of  appetite,  digestive  dis- 
turbances, lassitude,  and  sleeplessness.  Nose  bleed  is  a  peculiar  and 
rather  constant  feature.  The  temperature  and  pulse  gradually  rise 
until  by  the  end  of  five  to  seven  days  the  former  has  become  high, 
io3°F.  to  io4°F.  and  constant.  The  temperature  continues  thus 
through  the  second  week  during  which  a  gradual  stupor  and  occasional 
delirium,  diarrhoea,  and  enlargement  of  the  spleen  occur.  The  pulse  is 
often  dicrotic  and  there  is  a  rash  consisting  of  isolated  flattened  rose- 

*  Prepared  by  Edward  Fidlar. 


846    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

colored  macules  or  spots  which  may  be  few  or  numerous  and  occur  in 
successive  crops.  During  the  third  week  in  mild  cases  these  symptoms 
gradually  subside.  In  severer  forms  no  abatement  is  shown  and  com- 
plications are  liable  to  occur.  The  fourth  week  shows  beginning 
convalescence  in  the  typical  case. 

The  characteristic  pathological  findings  are  swelling  and  ulceration 
of  the  lymphoid  structures  of  the  lower  part  of  the  small  intestine  best 
seen  in  the  Peyer's  patches  of  the  ileum  just  above  the  ileo-cecal  valve. 
The  mesenteric  glands  and  spleen  are  hyperaemic.  Parenchymatous 
degenerations  more  or  less  severe  may  be  found  in  other  organs.  The 
characteristic  histological  feature  is  the  crowding  of  the  lymph  spaces 
by  proliferated  endothelial  cells. 

Perforation  and  hemorrhage  of  the  bowel,  peritonitis,  myocarditis, 
thrombosis,  etc.,  render  typhoid  fever  a  dangerous  disease.  The  fatality 
varies  considerably;  at  one  time  estimated  at  25  per  cent,  it  has  been 
brought  down  to  10  to  15  per  cent  by  modern  methods  of  treatment 
and  has  been  given  in  Minnesota  as  low  as  4  per  cent. 

Eberth  found  the  organism  in  1880  by  the  examination  of  the 
mesenteric  glands  and  spleen  of  fatal  cases.  GafTky  cultivated  it  in 
1884.  The  causal  relationship  has  been  a  matter  of  gradual  acceptance 
through  evidence  furnished  by  the  study  of  such  immunity  processes 
as  agglutination,  bacteriolysis,  and  complement  deviation,  and  finally 
by  the  high  percentage  of  positive  blood  cultures.  Conclusive  evidence 
is  afforded  by  the  development  of  typhoid  fever  following  the  ingestion 
of  pure  cultures  with  suicidal  intent. 

The  agglutination  reaction  of  Gruber  and  Widal  is  universally 
employed  in  diagnostic  laboratories.  The  blood  serum  of  typhoid 
patients,  after  a  certain  period  of  the  disease,  will  cause  a  characteristic 
clumping  of  the  bacilli  when  mixed  with  pure  cultures.  The  fresh 
serum  from  a  clot  may  be  used,  or,  more  conveniently,  dried  blood 
from  which  a  watery  extract  can  be  made.  In  positive  cases  the  reac- 
tion is  present  in  at  least  the  one-fiftieth  dilution  and  usually  in  the  one- 
hundredth  or  higher  dilutions.  The  culture  employed  should  be 
eighteen  to  twenty-four  hours  old;  it  should  be  freely  agglutinable  and 
show  no  artificial  clumping,  characters  not  possessed  by  all  strains, 
especially  those  recently  isolated.  Cultures  killed  by  a  small  per- 
centage of  carbolic  acid  have  been  recommended  for  constancy  in  place 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      847 

of  the  living  organisms.  When  the  microscopic  method  is  used  the 
reaction  should  be  distinct  in  about  one  hour. 

Owing  to  the  extensive  adoption  of  inoculation  against  typhoid  and 
paratyphoid  fevers,  the  problem  of  exact  diagnosis  has  become  more 
difficult.  If  culture  fails  to  reveal  the  exciting  organism  it  becomes 
necessary  to  take  a  series  of  at  least  three  agglutination  tests  at  inter- 
vals of  two  or  three  days  in  order  to  determine  the  comparative  curves 
of  agglutinin  for  B.  typhosus,  B.  paratyphosus  A.  and  B.  paratyphosus  B. 
For  this  purpose  Dreyer's  technique  has  been  largely  used. 

An  immune  body  capable  of  binding  complement  in  the  presence 
of  typhoid  antigen  is  said  to  occur  in  typhoid  sera  before  the  agglutina- 
tive property  appears. 

The  detection  of  the  typhoid  bacillus  in  the  circulating  blood  has 
been  very  widely  successful  and  furnishes  the  best  support  for 
the  diagnosis  of  the  disease.  While  blood  culture  may  be  hardly 
practical  in  public  health  laboratories,  it  has  become  a  routine  measure 
in  the  modern  hospital.  Blood  is  taken  aseptically  from  a  vein  and 
about  i  to  5  c.c.  is  introduced  into  culture  media,  of  which  fluid  media 
containing  ox-bile  and  agar  plating  media  containing  glucose  have  been 
most  strongly  recommended.  The  fluid  media  are  used  in  100  to  500 
c.c.  amounts,  which  serves  to  dilute  the  antibacterial  properties  of  the 
blood  while  the  bile  acts  as  an  anticoagulant  and  possibly  also  as  an 
antibactericidal  measure.  Plating  lessens  the  diffusion  of  the  anti- 
bacterial properties  and  thus  favors  growth. 

The  urine  and  faeces  have  sometimes  to  be  examined  for  the  presence 
of  B.  typhosus.  It  then  becomes  necessary  to  differentiate  the  colonies 
of  this  bacillus  from  those  of  the  colon  group.  For  this  purpose  many 
special  media  have  been  devised,  some  depending  on  the  motility  of  the 
typhoid  bacillus  to  form  a  different  shaped  colony  in  suitable  soft 
media,  others  based  on  the  fact  that  some  substances  such  as  fuchsin, 
crystal  violet,  malachite  green,  etc.,  inhibit  the  growth  of  associated 
organisms  while  permitting  the  typhoid  bacillus  to  develop  more  or  less 
luxuriantly. 

As  found  in  pure  cultures,  the  bacillus  is  about  i/x  to  3.5*1  in  length  and  0.5/1  to 
O.SM  in  width  (Fig.  178).  Filaments  are  sometimes  found  several  times  the  length 
of  the  single  organism.  It  is  quite  regular  in  shape,  straight  with  rounded  ends. 
The  bacilli  usually  occur  singly;  occasionally  two  may  be  attached  end  to  end  for  a 
short  time.  There  are  ten  to  fourteen  comparatively  stout  flagella  about  two  or 


848   MICROBIOLOGY   OF  DISEASES   OF  MAN  AND   DOMESTIC   ANIMALS 

three  times  the  length  of  the  organism  peritrichic  in  their  arrangement.  There 
are  no  capsules  and  no  spores.  They  stain  with  all  aniline  dyes,  and  not  infre- 
quently exhibit  more  deeply  staining  areas  at  the  poles.  They  are  Gram-negative. 
Biological  and  biochemical  characters: — The  minimum  temperature  is  about  10°, 
the  optimum  37°,  maximum  40  to  41°.  It  is  aerobic  and  facultatively  anaerobic. 
The  slight  preference  for  oxygen  is  probably  of  little  account  when  such  sugars  as 
glucose  are  present.  The  bacillus  is  not  very  sensitive  to  the  reaction  of  media  and 
will  grow  in  the  presence  of  either  slightly  alkaline  or  acid  reaction.  Alkaline 


FIG.  178. — Bacillus  of  typhoid  fever.     X  1000.     (After  Williams.} 

substances  are  produced  from  peptone.  Acid  is  formed  from  dextrose,  levulose, 
galactose,  mannit,  maltose,  and  dextrin.  Lactose  and  saccharose  remain  un- 
changed. Gas  is  never  formed.  It  is  the  rule  that  the  Bacillus  typhosus  does  not 
form  indol;  certain  strains,  however,  form  a  trace.  The  toxins  of  the  bacillus  have 
been  very  widely  studied  and  several  different  opinions  are  held  with  regard  to 
their  nature.  Most  evidence  supports  the  idea  that  the  poisons  are  only  set  free 
by  the  destruction  of  the  bacterial  bodies.  This  may  be  brought  about  experi- 
mentally by  various  means  such  as  the  use  of  lytic  or  bactericidal  sera,  by  the 
disintegration  occurring  in  old  cultures,  by  extraction  under  great  pressures,  by 
triturating  after  freezing  in  liquid  air  and  by  emulsifying  cultures,  sterilizing  by 
heat,  then  extracting  with  salt  solution.  These  endotoxins,  however  obtained  out- 
side of  the  host,  have  been  found  to  produce  by  injection  into  animals  only  lytic 
and  bactericidal  sera  and  not  an  antitoxin.  More  recently,  however,  some  ob- 
servers claim  to  have  shown  in  comparatively  young  cultures  the  presence  of  a  sub- 
stance which  upon  injection  into  animals  yields  an  antitoxin  and  thus  comports  itself 


MICROBIAL  DISEASES    OF   MAN  AND   DOMESTIC  ANIMALS      849 

after  the  manner  of  a  true  diffusible  or  soluble  toxin.  Agar  streak  cultures  show  an 
abundant  filiform  whitish  or  bluish-gray  translucent  growth  with  no  special  char- 
acteristics. Broth  is  uniformly  and  moderately  clouded  and  only  occasionally  a 
delicate  pellicle  may  develop.  Gelatin  colonies  are  bluish  white  in  color,  trans- 
parent and  with  somewhat  notched  margins.  Stab  cultures  show  more  growth  at 
the  surface,  while  in  the  depth  the  growth  is  filiform  and  less  abundant.  The 
medium  is  not  liquefied.  Milk  is  not  coagulated.  In  litmus  milk  there  may  be  a 
trace  of  acid  formed  at  first,  followed  by  a  return  to  neutral  or  very  slightly  alkaline 
reaction.  Potato  was  at  one  time  considered  a  very  valuable  differential  medium. 
The  growth  of  the  bacillus  upon  it  is  quite  abundant,  glistening,  but  invisible, 
when  the  potato  is  acid.  A  more  alkaline  reaction  allows  a  rather  heavy  yellowish 
growth  indistinguishable  from  B.  coli.  Special  media  are  used  in  the  cultivation 
of  the  typhoid  bacillus,  chiefly  for  differential  purposes.  The  cultural  features  on 
these  do  not  show  sufficiently  striking  characters  to  make  it  worth  while  to  review 
the  many  that  have  been  devised.  Specific  agglutinating  and  bacteriolytic  sera 
as  well  as  the  complement  binding  reaction  are  valuable  aids  in  identifying  the 
bacillus.  Resistance  to  heat  and  light  is  not  different  from  that  of  the  average  non- 
spore-bearing  species.  Its  thermal  death-point  is  about  56°  for  ten  minutes,  60° 
for  one  minute.  Exceptionally  resistant  forms  have  been  found  alive  in  ice  after 
three  months.  Sometimes  the  bacilli  will  remain  viable  for  a  month  after  drying. 
At  other  times  they  die  out  rapidly.  They  have  been  found  to  be  viable  for  ten 
days  in  distilled  water,  while  pure  sodium  chloride  dissolved  exerted  an  unfavorable 
influence.  In  faeces  the  length  of  life  is  from  a  few  hours  to  several  days,  or  even  as 
high  as  five  months  in  winter.  Their  life  in  privies  and  cesspools  is  ordinarily 
brief  but  has  been  found  to  extend  for  thirty  days.  Of  the  non-spore-formers 
the  bacillus  appears  to  be  rather  more  resistant  than  the  average  but  succumbs 
within  five  minutes  to  i  :  5000  mercuric  chloride  or  5  per  cent  phenol. 


The  organism  enters  the  body  through  the  mouth  by  means  of 
infected  fingers,  food,  milk,  and  water,  etc. 

On  reaching  the  intestine  the  organism  probably  propagates  to  some 
extent  before  penetrating  the  intestinal  mucosa.  It  enters  into  the 
blood  stream  and  is  disseminated  throughout  the  body.  According 
to  the  endotoxin  theory  it  must  slowly  be  dissolved  by  the  lytic  sub- 
stances which  have  been  gradually  accumulating  in  response  to  the 
primary  intoxication.- 

The  organisms  have  been  cultivated  from  the  rose  spots  and  have 
been  found  in  vomit  without  the  presence  of  blood,  and  in  sputum. 
Typhoid  meningitis  and  osteitis  occur  occasionally.  At  autopsy  the 
'spleen  and  gall-bladder  yield  the  highest  number  of  positive  cultures. 
It  is  of  interest  to  note  too  that  while  the  highest  percentage  (89-90 
per  cent)  of  positive  blood  cultures  occurs  in  the  first  week  and  the 


850   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

percentage  diminishes  from  then  on,  the  number  of  positive  findings 
in  the  faeces,  on  tLj  other  hand,  runs  in  the  opposite  direction. 

Generally  speaking,  one  attack  confers  immunity.  Upon  what 
antibodies  immunity  and  recovery  depend  is  a  matter  of  controversy. 

The  elimination  of  the  bacilli  from  the  body  will  largely  depend  upon 
the  stage  of  the  disease,  since  the  blood,  especially  early  in  the  illness, 
practically  always  contains  the  specific  organism;  epistaxis  is  not  an 
unimportant  feature  as  a  possible  means  of  disseminating  the  germs. 
The  bacilli  can  also  escape  in  the  faeces,  urine,  sputum,  and  vomit. 

In  the  control  of  this  disease  the  best  place  to  begin  is  at  the  bedside. 
Disinfection  of  all  excreta  and  of  everything  which  comes  into  con- 
tact with  the  patient  should  be  rigorously  carried  out  and  in  the  case  of 
the  faeces  and  urine  should  ideally  be  continued  until  examination  can 
be  made  showing  absence  of  the  organism.  It  has  been  estimated  that 
as  high  as  5  per  cent  of  convalescents  continue  to  excrete  living  typhoid 
bacilli  for  varying  periods  from  months  to  years  after  the  disease;  the 
longest  time  noted  has  been  forty-six  years. 

The  recognition  of  typhoid  carriers  will  depend  absolutely  on  the 
finding  of  the  specific  germ  in  the  faeces  or  urine  as  the  case  may  be. 
Where  there  are  large  numbers  of  suspects,  the  opsonic  index  is  claimed 
to  be  an  aid  in  exclusion  of  the  improbable  ones,  as  well  as  the  agglutinin 
reaction. 

In  a  general  way,  prompt  recognition  of  the  source  of  infection  such 
as  milk,  polluted  water,  bacilli-carriers,  etc.,  together  with  instruction 
of  the  individual  and  the  public  are  often  effective  in  limiting  and  end- 
ing an  epidemic. 

While  a  great  many  sera  have  been  used  therapeutically  with  some 
success,  prophylaxis  promises  more  where  it  can  be  widely  employed  as 
in  armies  and  navies.  The  artificial  immunity  is  brought  about  by  in- 
jection of  dead  cultures.  A  difference  of  about  25  per  cent  has  been 
noted  between  the  percentage  of  cases  in  vaccinated  and  unvaccinated 
persons  in  civil  life. 

In  the  United  States  Army,  the  establishment  of  compulsory  anti- 
typhoid inoculation  demonstrated  most  remarkably  favorable  results 
during  the  year  1913.  Amongst  90,646  men,  both  American  and  native 
troops,  only  three  cases  of  typhoid  fever  occurred  and  two  of  these  were 
infected  before  enlistment;  there  were  no  deaths.  When  comparison  was 
made  with  the  best  results  obtained  in  the  army  from  sanitary  measures 


MICROBIAL  DISEASES    OF   MAN  AND   DOMESTIC   ANIMALS      851 

alone  without  the  vaccination,  Major  Russell  estimated  that  in  1913  there 
was  "only  one  one-hundred-and-sixty-seventh  of  the  loss  of  time  from 
duty  because  of  typhoid  fever." 

Antityphoid  inoculation  was  of  inestimable  value  in  the  great  war. 
Exact  figures  cannot  be  given,  but  as  an  indication  of  what  might  have 
occurred  without  it,  the  fact  has  been  mentioned  that  a  small  portion 
of  the  French  army  which  in  the  early  critical  days  had  to  be  hurried 
to  the  front  without  inoculation,  developed  as  many  cases  of  typhoid 
fever  as  had  occurred  in  the  British  army  during  the  whole  Boer  war. 

ASIATIC  CHOLERA* 
Microspira  comma 

The  disease  is  endemic  in  parts  of  India  whence  epidemics  have 
spread  throughout  the  world.  America  has  been  visited  by  several 
epidemics  and  at  the  sea  ports  more  frequently,  chiefly  New  Orleans. 

The  disease  occurs  naturally  only  in  man.  The  incubation  period 
is  from  part  of  a  day  to  ten  days,  usually  about  three  days. 

In  its  most  characteristic  form  the  disease  begins  with  few  or  no 
prodromata.  It  is  marked  by  fever,  sudden  onset  of  purging  and 
vomiting  followed  by  cramps  and  severe  depression.  Evacuations 
finally  become  almost  a  colorless  liquid,  "rice-water  stools."  The 
cramps  may  occur  in  the  whole  muscular  system  most  frequently  in 
the  legs  and  are  often  extremely  painful.  A  stage  of  complete  collapse 
finally  occurs.  There  are,  however,  many  variations  from  these 
typical  cases.  The  mortality  is  usually  given  at  from  45  to  50  per 
cent. 

After  death  there  are  found  extensive  acute  degenerative  changes  in 
the  kidneys;  the  gastro-intestinal  tract  shows  marked  changes  in  the 
lining  membrane  which  may  be  necrotic,  sodden  and  in  some  places 
stripped  away. 

The  cholera  vibrios  may  sometimes  be  seen  in  enormous  numbers  in  smears  from 
typical  stools.  For  a  positive  diagnosis,  however,  the  organism  must  be  cultivated. 
The  usual  method  is  to  inoculate  a  i  per  cent  peptone  solution  from  the  stool,  in- 
cubating at  37°  for  from  four  to  eight  hours  and  sowing  plates  from  the  very  surface 
of  the  liquid,  either  of  gelatin  or  alkaline  agar  or  both.  The  vibrios  are  3/i  to  5/11 
long  by  about  o.^p.  wide,  and  are  curved  slightly  like  a  comma  or  sometimes  in  a 
half  circle.  These  comma  forms  are  best  seen  in  broth  cultures.  The  ends 
are  usually  rounded.  In  young  cultures  the  organisms  are  usually  arranged 

*  Prepared  by  Edward  Fidlar. 


852    MICROBIOLOGY   OF   DISEASES   OF   MAN  -AND   DOMESTIC   ANIMALS 

singly,  occasionally  two  may  be  found  end  to  end  in  the  form  of  an  "  S."  There  is 
no  capsule,  and  no  Spdre  formation.  There  is  a  single  terminal  flagellum,  and  the 
organism  is  exceedingly  motile.  Does  not  stain  as  readily  with  the  ordinary  aniline 
dyes  as  many  other  bacteria.  Fuchsin  gives  the  best  result.  It  is  Gram-negative. 
The  optimum  temperature  for  growth  is  37°  with  a  minimum  of  8°  and  a  maximum 
of  42°.  Plain  agar — moist,  shining,  grayish  yellow,  and  rather  thin  and  transparent 
as  compared  with  the  colon  type  of  colony.  A  rapid  growth  takes  place  in  broth, 
causing  a  uniform  clouding  with  a  more  or  less  well-developed  pellicle.  In  gelatin 
plates  colonies  are  visible  in  twenty-four  hours  and  are  round,  even,  and  yellowish 
white,  later  they  become  irregular  and  their  surface  presents  fine  refractile  granules; 
within  forty-eight  hours  the  colonies  are  found  to  be  sinking  into  a  small  round  pit 
due  to  liquefaction  of  the  medium  (Fig.  180).  Concentric  rings  may  appear  as 
liquefaction  progresses  from  day  to  day.  In  old  cultures  the  liquefaction  assumes  a 


FIG.  179. — Microspira  comma.     X  1000.     (After  Williams) 

funnel  or  turnip  shape  with  an  air  bubble  at  the  surface  due  to  evaporation.  Growth 
in  milk  occurs  without  any  visible  change  in  the  medium.  At  37°,  on  potato,  an 
abundant  moist  brownish  growth.  Blood  serum  is  liquefied  rapidly.  The  vibrios 
prefer  the  presence  of  oxygen,  yet  it  is  probable  that  organisms  grow  under  practically 
anaerobic  conditions  in  the  intestine.  The  reaction  of  all  media  must  be  very  dis- 
tinctly alkaline  and  even  very  small  amounts  of  acid  are  inhibitive.  Neither  gas 
nor  acid  is  formed.  The  production  of  indol  and  the  formation  of  nitrites  from 
nitrates  occurs  regularly.  The  addition  of  sulphuric  acid  is  sufficient  to  give  the 
nitroso-indol  reaction,  which  from  its  association  with  this  bacterium  has  been  called 
the  cholera  red  reaction.  No  pigment  is  produced.  Majority  of  freshly  isolated 
cultures  have  haemolytic  powers.  It  is  generally  considered  that  there  is  only  an 
endotoxin,  but  it  is  strongly  asserted  by  some  that  a  soluble  toxin  is  formed.  Thermal 
death-points  are  60°  for  ten  minutes,  95°  to  100°  for  one  minute.  Vibrios  are 
quite  sensitive  to  low  temperature  and  at  most  have  been  found  viable  in  ice  only 
after  a  few  days.  The  vibrios  are  quite  susceptible  to  the  ordinary  disinfectants. 


MICROBIAL   DISEASES   OF   MAN  AND   DOMESTIC   ANIMALS      853 

The  cholera  organism  gains  entrance  through  the  mouth. 

Having  succeeded  in  passing  the  acid  secretions  of  the  stomach  the 
vibrios  probably  develop  with  great  rapidity  in  the  small  intestine. 

The  peculiar  conditions  favorable  to  the  development  of  the 
organism  in  the  intestine  are  unknown.  A  previous  gastro-intestinal 
disturbance  is  probably  necessary  even  though  slight. 

The  organisms  have  rarely  been  demonstrated  in  blood  cultures. 
The  gall-bladder  gives  the  highest  percentage  of  positive  cultures. 


FIG.  1 80. — Micros pira  comma.  Colonies  on  gelatin  plates,  a,  Twenty-four  hours 
old;  b,  thirty  hours  old;  c,  forty-eight  hours  old.  (After  Fraenkel  and  Pfeifer 
from  Williams.} 


Highly  ly tic  and  agglutinating  sera  can  be  developed  experimentally, 
but  little  or  no  antitoxic  power  can  be  demonstrated. 

Protective  inoculation  has  shown  considerably  more  encouraging 
results  than  serum  therapy. 

The  cholera  vibrios  are  eliminated  in  the  discharges.  Water  and 
uncooked  food  becoming  contaminated  with  cholera  excreta  are  the 
chief  means  by  which  the  epidemic  is  spread,  so  that  its  epidemiology 
is  similar  to  that  of  typhoid  fever. 


854  MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

MICROBIA*,  DISEASES  AS  YET  UNCLASSIFIED* 

SCARLET   FEVER,    MEASLES,    GERMAN   MEASLES,  DUKE'S    DISEASE, 
SMALLPOX,  CHICKENPOX,  MUMPS f 

These  diseases  constitute  a  group  the  actual  biological  causes  of 
which  are  unknown,  yet  which  show  analogies  to  diseases  the  causes  of 
which  are  known,  so  close  as  to  make  tenable  the  hypothesis  that  they 
are  due  to  similar  causes. 

Mumps  is  in  a  class  by  itself,  its  characteristics,  well  known  to  the 
laity,  marking  it  off  from  the  others  sharply.  Like  the  others  it  is 
infectious;  it  is  derived  only  from  a  preceding  case;  it  has  a  more  or 
less  definite  incubation  period  (i.e.,  an  interval  between  the  date  of 
infection  and  the  first  development  of  symptoms,  during  which  ordinary 
health  is  enjoyed),  and  a  prodromal  stage  (i.e.,  a  period  in  which  fever, 
headache,  and  other  more  or  less  marked  constitutional  symptoms  exist 
without  any  marked  characteristic  symptom).  Then  appears  the 
swelling  of  the  parotid  salivary  glands  just  in  front  of  the  ears  with 
some  pain.  The  symptoms  usually  amend  after  a  few  days  and  the 
patient  goes  on  to  full  recovery.  There  is  no  rash  nor  any  great 
disturbance  of  the  intestinal  tract  or  internal  organs  as  a  rule, 
although  metastases,  affecting  the  mammae,  ovaries  or  testicles  de- 
velop at  times;  and  secondary  complications  sometimes  are  found. 

Smallpox  and  chickenpox  together  form  a  group  quite  often  confused 
clinically,  especially  in  the  early  stages  and  especially  when  smallpox 
is  prevalent  in  mild  form.  They  have  incubation  periods,  approxi- 
mating about  twelve  days,  in  smallpox  varying  little  from  this  period, 
in  chickenpox  varying  widely  from  it.  Smallpox  has  rather  severe 
prodromes,  backache,  headache,  fever,  and  sore  throat,  the  rash  appear- 
ing on  the  third  or  fourth  day.  Chickenpox  usually  has  light  or  no 
prodromes,  the  rash  appearing  on  the  same  day  or  within  twenty- 
four  hours,  as  a  rule.  In  both  diseases  the  face,  chest,  back,  arms, 
hands,  legs,  and  feet  are  likely  to  show  eruption,  but  chickenpox  tends 
to  show  the  greatest  number  of  spots  "  under  cover,"  i.e.,  on  the  parts 
usually  covered  by  clothing,  while  smallpox  tends  to  show  the  majority 
upon  the  face,  neck,  arms,  wrists,  hands,  legs  and  feet  rather  than  on  the 
body.  The  skin  lesions  themselves  differ  very  markedly,  the  typical 

*  Arranged  alphabetically  except  group  of  diseases  placed  first. 
f  Prepared  by  H.  W.  Hill. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      855 

lesions  of  chickenpox  being  superficial,  thin  walled,  high,  rounded, 
and  filled  with  clear  liquid,  those  of  smallpox  being  deep  seated,  tense, 
opaque,  with  a  tough  covering  of  epithelium.  There  are  many  other 
points  of  distinction,  and  any  one  familiar  with  the  two  diseases  can 
hardly  fall  into  error  when  dealing  with  typical  cases  at  whatever  stage 
they  are  encountered.  To  the  layman's  eye,  however,  the  two  are 
often  indistinguishable. 

Scarlet  fever,  measles,  German  measles,  and  Duke's  disease  are  often 
likewise  confused  by  the  laity  and  even  by  physicians  who  have  not 
had  opportunities  for  extensive  study. 

German  measles  is  clinically  related  to  true  measles  somewhat  as 
chickenpox  is  to  smallpox,  i.e.,  they  are  wholly  distinct  diseases  yet 
show  characteristics  easily  confused  on  superficial  consideration. 
Duke's  disease  is  perhaps  not  a  distinct  entity;  much  has  been  said  on 
this  point  and  a  satisfactory  decision  will  probably  never  be  reached 
until  the  causative  agents  have  been  found.  It  may  be  described 
briefly  for  clinical  purposes  as  a  variety  of  German  measles  having  a 
scarlatiniform  instead  of  a  measly  rash. 

Scarlet  fever  has  an  average  incubation  period  of  about  five  days, 
or  perhaps  sometimes  less.  The  prodromes  are  those  usual  to  all 
these  infections — headache,  fever,  and  sore  throat,  but  the  latter 
is  especially  severe.  Within  twenty-four  hours  the  rash  appears  usually 
on  the  chest  first,  a  bright  scarlet  superficial  punctate  flush,  extending 
rapidly  over  the  body. 

In  measles  the  incubation  period  is  longer,  averaging  nine  or  ten 
days,  almost  without  any  variation.  The  prodromes,  headache,  fever, 
and  sore  throat,  are  accompanied  by  very  marked  coryza  and  photo- 
phobia, catarrh  and  "cold  on  the  chest." 

The  rash  appears  about  the  fourth  day,  appearing  on  the  face  and 
back  but  rapidly  extending.  It  is  darker,  bluer,  and  deeper  than  the 
scarlet  fever  rash  and  unlike  the  latter  is  palpable.  Koplik's  spots 
appear  on  the  buccal  membrane  early  in  the  disease. 

In  German  measles  the  prodromes  are  so  indefinite  that  it  is  difficult 
to  determine  their  length;  very  commonly  the  rash  is  the  first  thing  no- 
ticed. It  appears  on  the  face,  chest,  back,  and  arms  as  a  light  sub- 
cuticular  mottling  (measles  type)  or  a  more  uniform  pink  flush  (scarla- 
tiniform or  Duke's  type) ;  with  this  rash  the  eyes  show  some  injection 
and  slight  photophobia  develops.  The  attack  passes  off  quickly, 
without  complications. 


856   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

CANINE  DISTEMPER* 

This  disease  (Maladie  des  jeunes  chiens;  Fr.)  is  so  widespread  that 
the  great  majority  of  adult  dogs  may  be  regarded  as  having  suffered 
from  an  attack  and  recovered.  It  is  practically  confined  to  very  young 
animals  and,  so  far  as  known,  no  species  except  dogs  are  susceptible. 
The  disease  is  attended  by  more  or  less  extensive  coryza  with  a  dis- 
charge from  the  eyes.  There  is  an  eruption  on  the  skin  and  frequently 
nervous  disorders  of  various  kinds.  The  animal  becomes  emaciated 
and  may  die  from  bronchial  pneumonia.  No  organism  has  been  fully 
accepted  as  the  cause  of  this  disease.  Carre  has  reported  that  he  has 
succeeded  in  passing  the  infectious  agent  in  nasal  discharges  through 
earthen  filters,  the  filtrate  reproducing  distemper  in  characteristic  form. 
Ferry  has  announced  the  discovery  of  an  organism  as  the  causal 
agent.  Some  attempts  have  been  made  to  produce  a  protective  serum. 

CATTLE  PLAGUE* 

This  disease  (rinderpest),  which  is  probably  the  severest  and  most 
contagious  of  all  cattle  diseases,  is  characterized  by  high  fever  and 
lesions  of  the  intestinal  tract.  It  does  not  exist  in  the  United  States 
but  is  found  in  Europe,  S.  Africa  and  Asia.  Extensive  outbreaks 
have  occurred  in  the  Philippine  Islands.  The  cause  of  cattle  plague  has 
never  been  isolated  and  the  indications  are  that  it  is  caused  by  a 
filtrable  microorganism.  Cattle  plague  was  the  first  disease  in  which 
the  process  of  "  hyperimmunization "  was  practiced.  Immune  cattle 
receive  massive  injections  of  blood  from  diseased  cattle.  After  this 
treatment  the  blood  serum  of  the  immune  is  used  to  protect  non- 
immunes.  Enormous  quantities  of  this  serum  are  prepared  and 
applied  yearly  by  the  British  government  in  India. 

CONTAGIOUS  BOVINE  PLEURO-PNEUMQNIA* 

This  disease  affects  cattle  only;  it  is  highly  infectious  and  produces 
an  inflammation  of  the  lungs  and  pleural  membranes.  Thirty  years 
ago  bovine  pleuro-pneumonia  was  quite  prevalent  in  the  United  State? 
but  has  since  been  eradicated  through  the  efforts  of  the  Federal  Bureau 
of  Animal  Industry  in  cooperation  with  State  authorities.  It  still 
exists  in  European  countries. 

*  Prepared  by  M.  Dorset. 


MICROBIAL  DISEASES    OF  MAN  AND   DOMESTIC   ANIMALS      857 

The  microorganism  of  bovine  pleuro-pneumonia  is  generally  classed 
among  the  filtrable  viruses,  though  unlike  some  organisms  of  that 
class  it  has  been  cultivated  artificially  and  is  just  visible  at  a  magni- 
fication of  2,000  diameters.  The  artificial  cultivation  of  this  virus  was 
accomplished  by  Roux  and  Nocard  through  the  use  of  the  very  in- 
genious "collodion  sac  method."  A  small  amount  of  virus  from  a 
diseased  cow  was  placed  within  a  small  thin- walled  sac  of  collodion; 
after  being  hermetically  sealed  the  sac  was  placed  in  the  peritoneal 
cavity  of  a  rabbit  where  it  remained  for  several  weeks.  At  the  end  of 
this  time  the  unbroken  sac  was  removed  and  the  previously  clear  fluid 
within  was  found  to  be  slightly  opalescent.  Microscopic  examination 
revealed  numberless  minute  motile  bodies  so  small,  however,  that  their 
exact  form  could  not  be  determined.  Later  the  organism  was  suc- 
cessfully cultivated  outside  of  the  animal  body  in  a  specially  prepared 
bouillon.  These  cultures  produced  the  disease  when  inoculated  Into 
susceptible  cattle.  When  the  virus  is  diluted  it  will  pass  through  the 
Berkefeld  and  Chamberland  F  cylinders,  but  not  through  the  Chamber- 
land  B  cylinder. 

COWPOX,    HORSEPOX,   AND   SHEEPPOX*  . 

Variola  refers  to  a  condition  or  disease  in  man  and  animals,  charac- 
terized by  fever  and  the  appearance  of  skin  eruptions  which  succes- 
sively assume  the  form  of  papules,  vesicles  and  pustules.  The  disease 
is  frequently  found  in  the  human  species  (smallpox),  cattle  (variola 
vaccinia,  cowpox),  horses  (variola  equince,  horsepox)  and  sheep  (variola 
ovina,  sheeppox).  It  is  possible  that  some  other  species  may  be 
susceptible. 

On  account  of  the  fact  that  vaccination  of  man  with  virus  from  cases 
of  cowpox  affords  remarkable  protection  against  smallpox,  it  appears 
reasonable  to  believe  that  cowpox  virus  or  smallpox  vaccine  is  a 
modified  form  of  smallpox  virus.  This  fact,  together  with  the  occa- 
sional positive  results  of  various  experiments  in  which  other  species 
of  animals  have  at  times  evidenced  susceptibility  to  cowpox  virus, 
strongly  suggests  the  possible  etiological  relationship  of  the  diseases  in 
different  species  to  each  other  and  to  smallpox  in  man.  However, 
conclusive  proof  supporting  this  suggested  relationship  does  not  exist. 
The  specific  causative  factor  of  smallpox  or  of  cowpox  is  not  known. 

*  Prepared  by  W.  E.  King. 


858   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

Cowpox  is  a  very  common  disease,  perhaps  having  been  prevalent 
in  England  and  Europe  for  centuries.  Its  presence  has  frequently 
been  observed  in  various  countries  since  1796  when  Jenner  contributed 
to  the  world  his  important  discovery  relative  to  smallpox  vaccination. 

Many  attempts  have  been  made  to  isolate  the  causative 
factor  of  cowpox.  Early  investigators  frequently  secured  mixed  and 
pure  cultures  of  various  organisms,  including  different  species  of 
micrococci,  streptococci  and  bacilli  from  vaccine  lymph.  None  of 
these  organisms  were  peculiar  to  the  virus,  and  at  present  there  exists 
no  definite  evidence  that  the  infectious  agent  of  vaccine  lymph  is  of 
bacterial  nature.  Pfeiffer,  Guanieri,  Plimmer,  Councilman,  Mac- 
Grath,  Brinckerhoff  and  others,  after  observing  the  presence  of  apparent 
cellular  elements,  or  relatively  large  flattened  bodies  in  vaccine  lymph, 
have  suggested  the  possible  protozoan  nature  of  the  causative  agent. 
Attempts  have  been  made,  with  more  or  less  success,  to  cultivate  these 
bodies  in  collodion  capsules  in  the  peritoneal  cavities  of  experimental 
animals.  According  to  some  investigators  the  virus  has  been  passed 
through  a  Chamberland  filter.  The  failure  to  discover  the  causative 
factc-r,  according  to  the  present  methods,  may  be  due  to  the  inability 
of  microbiologists  to  cultivate  or  stain  the  specific  agent. 

Cowpox  is  characterized  by  eruptions  which  usually  occur  on  the 
skin  of  the  teats  and  udder.  The  material  contained  in  these  pustules 
is  transferred  to  other  animals  by  the  hands  of  the  milker  and  through 
other  possible  means  of  dissemination.  The  chief  channel  of  infection 
appears  to  be  through  an  abrasion  in  the  skin.  The  period  of  incuba- 
tion of  cowpox  is  about  two  days.  The  virus  possesses  relatively  weak 
resistance  to  heat,  light  and  chemicals.  The  control  of  the  disease 
depends  chiefly  upon  precautions  relative  to  the  transmission  of  the 
virus  on  the  hands  of  the  milker  from  infected  to  healthy  cows. 

Horsepox  may  be  diagnosed  by  the  appearance  of  the  characteristic 
pustules  usually  upon  the  skin,  nasal  mucosa  and  buccal  membrane. 

Sheeppox  is  characterized  by  the  presence  of  the  typical  skin 
eruptions,  following  a  rise  of  temperature. 

DENGUE* 

This  disease  (break-bone  fever)  of  man  occurs  in  all  parts  of  the 
world.  It  is  characterized  by  a  sudden  attack,  intense  prostration 

*  Prepared  by  M.  Dorset. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      859 

and  severe  pains  in  the  muscles  and  joints.  The  fever  during  the  attack 
shows  a  characteristic  curve.  There  is  a  sudden  rise  of  and  main- 
tained temperature  for  several  days.  Then  a  remission  and  a  second 
rise  of  temperature  which  is  less  than  the  first. 

Our  knowledge  of  the  cause  of  this  disease  rests  chiefly  upon 
researches  of  Ashburn  and  Craig.*  These  authors  conclude  that 
dengue  is  not  contagious  in  the  ordinary  sense  but  that  it  is  transmitted 
through  the  bite  of  the  mosquito  (Culex  fatigans).  No  visible  organ- 
ism could  be  demonstrated  in  either  fresh  or  stained  specimens  of 
blood  from  patients  affected  with  dengue  although  such  blood  was 
capable  of  producing  a  typical  attack  of  dengue  when  inoculated 
intravenously  into  healthy  men.  The  authors  likewise  show  that 
blood  from  a  case  of  dengue  retained  its  infectiveness  after  passage 
through  a  filter  made  of  diatomaceous  earth. 

FOOT-AND-MOUTH  DISEASE! 

Foot-and-mouth  disease  is  primarily  a  disease  of  cattle,  though 
the  other  domestic  animals  and  man  may  be  attacked.  The  disease 
is  very  contagious  and  is  characterized  by  the  eruption  of  vesicles  in 
the  mouths,  on  the  udders  and  on  the  skin  surrounding  the  hoofs  of 
cattle.  It  is  very  prevalent  in  European  countries.  There  have  been 
three  outbreaks  in  the  United  States  all  of  which  were  promptly  eradi- 
cated by  vigorous  repressive  measures  instituted  by  the  Federal 
authorities. 

The  cause  of  this  disease  is  an  invisible  microorganism  which  exists 
in  the  lymph  from  the  vesicles  which  form  in  the  mouths  and  on  the 
feet  of  cattle.  This  virus  has  never  been  cultivated  artificially.  It 
passes  through  the  Berkefeld  cylinder  but  not  through  the  finer-pored 
Kitasato  filters;  it  is  quickly  destroyed  by  formaldehyde,  carbolic  acid 
and  similar  disinfectants. 

The  disease  is  readily  transmitted  from  one  animal  to  another  by 
contact  and  the  contagion  may  persist  for  some  time  in  the  manure,  or 
straw  from  infected  stables.  The  milk  of  infected  cows  has  been  said 
to  produce  the  disease  in  children. 

Animals  which  recover  from  an  attack  remain  immune  for  a  short 
time  only;  it  is  therefore  not  surprising  that  no  satisfactory  means  of 
artificial  immunization  has  been  devised. 

*  Ashburn,  P.  M.  and  Craig,  C.  P.:  Jour.  Inf.  Dis.,  Vol,  IV,  p.  440,  1907. 
t  Prepared  by  M.  Dorset. 


86o  MICROBIOLOGY  OF  DISEASES  OF  MAN  AND  DOMESTIC  ANIMALS 

FOWL  PLAGUE* 

This  disease  (Hiihner  Pest;  Ger.:  Peste  aviaire;  Fr.)  of  fowls,  which 
is  to  be  distinguished  from  chicken  cholera,  is  not  known  in  the  United 
States,  but  has  caused  extensive  losses  of  fowls  in  Europe,  particularly 
in  Italy.  Affected  chickens  cease  eating,  the  feathers  become  ruffled 
and  the  comb  darker  in  color.  The  lesions  found  at  autopsy  are  not 
constant,  but  a  pericarditis  is  usually  seen.  There  may  be,  also,  con- 
gestion of  the  lungs,  liver,  and  kidneys.  The  intestinal  lesions  are  not 
as  marked  as  is  the  case  in  chicken  cholera. 

Fowl  plague  has  been  shown  to  be  due  to  an  invisible  microorgan- 
ism which  is  present  in  the  heart  blood  and  in  practically  all  of  the 
organs  of  the  body.  Most  fowls  are  susceptible;  guinea-pigs  and  mice 
are  refractory  to  the  disease.  The  virus  passes  through  Berkefeld 
and  Chamberland  F  cylinders;  it  is  quite  resistant  to  drying  but  is 
destroyed  by  an  exposure  of  half  an  hour  to  a  temperature  of  60°. 
Several  authorities  have  passed  the  filtered  virus  through  four  or  more 
hens  successively,  thus  demonstrating  positively  that  the  filtered  virus 
is  capable  of  multiplication. 

HOG  CHOLERA* 

The  first  recorded  outbreak  of  hog  cholera  in  the  United  States 
occurred  in  Ohio  in  the  year  1833  and  it  now  exists  in  practically  all 
sections  of  this  country.  Hog  cholera  is  most  prevalent  in  the  late 
summer  and  fall,  although  outbreaks  are  reported  at  all  seasons  of  the 
year.  All  races  of  hogs  are  susceptible  and  the  average  mortality  is 
about  80  per  cent.  In  the  United  States  alone  the  losses  from  hog 
cholera  are  estimated  to  average  at  least  $15,000,000  annually.  Hogs 
only  are  attacked.  This  disease  is  supposed  to  have  been  introduced 
into  the  United  States  through  the  importation  of  hogs  from  Europe, 
where  it  is  known  under  the  names  "swine  fever"  (Br.),  " schweinepest" 
(Ger.),  and  "peste  du  pore"  (Fr.). 

The  essential  features  of  hog  cholera  may  be  briefly  summarized  as 
follows:  Extreme  contagiousness.  Symptoms  of  severe  illness  accom- 
panied by  fever,  loss  of  appetite,  weakness  and  diarrhoea.  Haemor- 
rhagic  lesions  in  the  various  organs  and  lymphatic  glands  and  round 

*  Prepared  by  M.  Dorset. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC   ANIMALS      86 1 

button-like  ulcers  in  the  large  intestine.     Immunity   in  hogs   which 
recover. 

The  etiology  of  hog  cholera  has  long  been  the  subject  of  scientific 
controversy,  but  it  is  now  generally  acknowledged  that  the  cause  of 
this  disease  is  a  filtrable  microorganism  which  exists  in  the  blood, 


FIG.  181. — Haemorrhagic  points  on  kidneys  of  hog-cholera  hog.     (Original.) 

the  internal  organs,  and  the  urine  of  infected  hogs.  The  fact  that 
this  disease  is  caused  by  a  filtrable  microorganism  was  demonstrated 
as  follows:* 

The  blood  serum  of  hogs  infected  with  hog  cholera  acquired  in  the 
natural  way  is  very  infectious  for  non-immune  hogs,  the  disease  being 
readily  transmitted  by  the  subcutaneous  injection  of  small  amounts. 
The  disease,  produced  by  this  subcutaneous  injection  is  identical  in  all 
respects  with  the  disease  as  it  occurs  in  nature.  If,  now,  this  infectious 

*  Bulletin  72,  Bureau  of  Animal  Industry,  U.  S.  Dept.  Agriculture,  1905. 


862    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

serum  is  diluted  with  normal  salt  solution  or  with  ordinary  bouillon 
(i  to  10)  and  passed  through  either  a  Berkefeld  or  Chamberland 
filter,  the  filtrate,  though  free  from  all  visible  microorganisms,  still  re- 
tains the  power  to  produce  hog  cholera  by  subcutaneous  injection. 
The  disease  which  is  produced  in  this  manner  by  the  filtered  hog 
cholera  serum  is  identical  in  all  respects  with  the  disease  produced  by 
the  unfiltered  serum  and  also  with  the  disease  as  it  occurs  in  nature. 
The  hogs  which  receive  the  filtered  serum  present  the  symptoms  and 
lesions  of  hog  cholera.  The  disease  set  up  in  this  manner  is  very 
contagious  and  hogs  which  recover  from .  the  inoculation  of  filtered 
serum  are  thereafter  immune  against  hog  cholera.  By  repeated 
inoculation  and  filtration  this  virus  may  serve  to  infect  successively  a 
large  number  of  hogs. 

The  invisible  virus  of  hog  cholera,  in  view  of  its  ability  to  pass 
through  the  Chamberland  B  filter,  must  be  regarded  as  one  of  the 
smallest  of  the  filtrable  microorganisms.  It  has  never  been  cultivated 
artificially,  hence,  aside  from  its  disease-producing  qualities,  we  have 
little  knowledge  concerning  it.  We  do  know,  however,  that  the  virus 
is  quite  resistant  to  such  common  disinfectants  as  carbolic  acid  and 
bichloride  of  mercury  and  that  it  is  quickly  destroyed  by  a  3  per  cent 
solution  of  liquor  cresolis  compositus  (U.  S.  P.)  as  well  as  by  a  5  per 
cent  solution  of  antiformin.  When  preserved  in  sealed  glass  bulbs 
in  a  cool  dark  place,  the  virus  retains  its  activity  for  six  months  or 
longer.  Rabbits,  guinea-pigs,  and  other  small  animals  are  entirely 
insusceptible  to  inoculations  of  the  filtered  virus  in  amounts  which 
would  prove  fatal  to  hogs. 

The  virus  of  hog  cholera  is  known  to  be  thrown  off  from  the  body 
through  the  urine,  the  faeces  and  the  eye  and  nose  secretions.  There- 
fore any  agency  which  would  serve  to  carry  a  particle  of  dirt  from  in- 
fected hog  yards  might  be  the  means  of  disseminating  the  virus.  As 
many  sick  hogs  find  their  way  to  the  public  stock  yards  through  ship- 
ment by  rail,  all  stock  cars  and  stock  yards  are  to  be  regarded  as 
permanently  infected.  It  appears  to  be  impracticable  to  prevent  the 
spread  of  the  disease  by  methods  of  quarantine  and  disinfection  alone, 
owing  to  the  impossibility  of  enforcing  such  measures  thoroughly.  It 
has  recently  been  found  that  a  protective  serum  against. hog  cholera 
may  be  produced  by  "hyperimmunization."  The  process  consists  in 
giving  immune  hogs  large  doses  of  blood  taken  from  hogs  sick  of  hog 


MICROBIAL  DISEASES    OF   MAN   AND   DOMESTIC  ANIMALS      863 

cholera.  As  a  result  of  this  blood  treatment  their  serum  acquires  the 
power  to  protect  non-immunes.  Injections  of  serum  from  hyper- 
immunized  animals  confers  a  passive  immunity,  while  the  simultaneous 
injection  of  serum  with  a  small  amount  of  virus  produces  an  active 
immunity. 

BACILLUS  CHOLERA  Suis  (B.  suipestifer). — No  description  of  the 
etiology  of  hog  cholera  would  be  complete  without  a  reference  to  this 
bacterium  which  was  long  regarded  as  the  cause  of  hog  cholera.  It  is 
found  after  death  in  the  blood  and  organs  of  the. majority  of  hogs 
affected  with  hog  cholera  and  in  this  r61e  of  secondary  invader  it  no 
doubt  tends  to  increase  the  mortality  from  the  disease.  B.  cholera 
suis  is  a  small,  very  actively  motile,  non-spore-bearing  bacillus  with 
rounded  ends,  and  stains  readily  with  the  ordinary  aniline  dyes.  It 
does  not  stain  by  Gram's  method.  This  organism  is  easily  cultivated 
on  the  ordinary  media;  gelatin  is  not  liquefied;  milk  is  not  coagulated 
but  acquires  an  acid  reaction  at  first;  this  changes  after  a  week  or  more 
to  an  alkaline  reaction.  Gas  is  produced  in  bouillon  containing 
dextrose,  but  lactose  and  saccharose  are  not  affected.  Rabbits  and 
guinea  pigs  succumb  within  four  to  ten  days  to  small  doses  of  this 
organism.  Hogs  are  much  more  refractory.  It  is  only  after  the  ad- 
ministration of  large  doses  that  they  show  any  symptoms  of  illness 
following  subcutaneous  injections.  By  feeding  pure  cultures  of  B. 
cholera  suis  or  by  injecting  these  intravenously  a  considerable  number 
of  hogs  will  succumb  and  at  autopsy  present  lesions  which  correspond 
quite  closely  to  those  seen  in  naturally  acquired  cases  of  hog  cholera. 
There  are,  however,  certain  important  differences  between  the  disease 
produced  by  B.  cholera  suis  and  the  natural  disease  hog  cholera.  For 
example,  hogs  infected  with  B.  cholera  suis  do  not  transmit  the  disease 
to  other  hogs  by  contact.  The  blood  of  hogs  infected  with  B.  cholera 
suis  does  not  produce  disease  when  injected  subcutaneously  into 
other  hogs,  and,  in  addition,  hogs  which  recover  from  illness  produced 
by  injections  or  feedings  of  pure  cultures  of  B.  cholera  suis  have  no 
immunity  against  the  natural  disease  hog  cholera. 

HORSE  SICKNESS* 

This  disease  affects  the  equine  species  only  and  appears  to  be  con- 
fined to  South  Africa.  It  is  most  prevalent  in  summer  and  appears  to 

*  Prepared  by  M.  Dorset. 


864   MICROBIOLOGY   OF  DISEASES   OF   MAN  AND   DOMESTIC  ANIMALS 

be  transmitted  by  the  bite  of  an  insect,  as  it  is  not  contagious  but  may 
be  communicated  to  susceptible  horses  through  blood  inoculations. 
This  disease  manifests  itself  by  producing  severe  inflammatory  changes 
in  the  lungs  and  in  the  tissues  of  the  head  and  neck  and  is  attended 
by  a  high  mortality.  No  visible  organism  has  been  found  which  will 
produce  horse  sickness  and  as  McFadyean  and  Nocard  have  shown  that 
the  virus  is  capable  of  passing  through  the  finest  bacteria-proof  filters, 
this  disease  is  probably  caused  by  an  invisible  microorganism.  Blood 
containing  the  microorganisms  of  horse  sickness  may  be  kept  in  sealed 
bulbs  in  the  dark  at  room  temperature  for  more  than  two  years  without 
losing  its  infectiveness.  The  virus  is  quite  resistant  to  drying  and 
may  survive  heating  for  ten  minutes  at  a  temperature  of  75°. 

INFANTILE  PARALYSIS* 

As  indicated  by  its  name,  this  disease  (epidemic  poliomyelitis)  is 
usually  seen  in  children.  It  has  long  been  known  to  exist  in  both 
Europe  and  America,  occurring  generally  in  sporadic  form.  During 
the  last  decade,  however,  its  prevalence  has  greatly  increased  and  a 
number  of  well-defined  epidemics  have  been  reported.  Though  the 
character  of  this  malady  long  ago  led  to  the  belief  that  it  was  caused  by 
a  microorganism,  this  fact  was  not  definitely  proven  until  the  year  1909 
when  Lands teiner  and  Popper  in  Germany,  and  Straus  and  Huntoon 
and  Flexner  and  Lewis  in  the  United  States,  succeeded  in  transmitting 
the  infection  to  monkeys.  So  far  as  is  now  known,  none  of  the  lower 
animals  except  monkeys  are  susceptible. 

The  symptoms  and  effects  of  infantile  paralysis  are  extremely 
variable.  Paralysis  is  by  no  means  constant,  many  cases  being  very 
mild  and  thus  possibly  escaping  detection.  In  the  severer  forms  of  the 
disease  paralyses  of  various  types  and  degrees  are  seen.  When  recovery 
takes  place  the  paralysis  may  appear  to  improve  only  to  be  followed  by 
atrophy  of  certain  groups  of  muscles,  resulting  in  deformity  and  per- 
manent lameness.  These  effects  are  caused  by  the  destruction  of 
certain  nerve  centers  in  the  spinal  cord. 

As  stated  above,  the  microbial  origin  of  infantile  paralysis  was  first 
demonstrated  by  the  inoculation  of  monkeys,  Flexner  and  Lewis  having 
successfully  carried  the  infection  through  a  long  series  of  monkeys  by 
successive  intracranial  injections  of  an  emulsion  of  the  spinal  cord 

*  Prepared  by  M.  Dorset. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      865 

taken  from  infected  animals.  The  microorganism  passes  through  the 
Chamberland  and  Berkefeld  niters  with  little  or  no  loss  in  disease- 
producing  power.  "Flexner  and  Noguchi,  employing  the  technic 
previously  used  for  cultivating  pathogenic  spirochaetes,  have  succeeded 
in  obtaining  from  infected  tissues  cultures  of  a  minute  round  organism 
which  they  believe  to  be  the  cause  of  infantile  paralysis."  The  virus 
withstands  freezing  or  drying  for  long  periods  of  time  but  is  quickly 
destroyed  by  heating  at  a  temperature  of  50°.  It  is  likewise  quickly 
killed  by  the  ordinary  disinfectants.  Monkeys  may  be  infected  by  the 
subcutaneous,  intraperitoneal,  intravenous,  or  intracranial  injection 
of  material  from  an  infected  spinal  cord,  but  attempts  at  infection 
through  feeding  have  been  unsuccessful.  The  virus  appears  to  be 
eliminated  from  the  body  through  the  nasal  mucous  membranes. 

It  appears  probable  that  one  attack  of  the  disease  protects  from  a 
second  attack.  No  cases  of  a  second  attack  have  been  reported. 
Furthermore,  monkeys  which  have  recovered  from  the  infection  appear 
to  be  entirely  immune  as  shown  by  Flexner.  Active  immunity  in 
monkeys  has  been  established  by  repeated  infections  of  gradually 
increased  amounts  of  the  virus.  The  blood  of  human  beings  and  of 
monkeys  that  have  recovered  from  an  attack  of  the  disease  is  capable 
of  neutralizing  a  certain  amount  of  the  virus.  This  protective  quality 
of  the  blood  serum  may  be  increased  by  repeated  inoculations  of  virus, 
and  infection  in  monkeys  can  be  prevented  by  injecting  simultaneously 
the  virus  into  the  brain  and  the  serum  into  the  sub-arachnoid  space. 
The  serum  treatment  of  this  disease  is,  however,  not  developed  to  such 
a  state  that  it  can  be  regarded  as  of  practical  use. 

PELLAGRA* 

Pellagra  is  a  disease  of  man  characterized  by  the  annually  recurring 
manifestation,  each  spring  or  summer,  of  erythema  on  the  backs  of 
the  hands  and  forearms  and  sometimes  on  the  face  and  neck,  feet  and 
ankles,  coupled  with  digestive  disorder  and  more  or  less  well-marked 
mental  disturbances.  During  the  winter  the  signs  of  the  disease  usually 
disappear. 

At  present  there  are  two  main  groups  of  theories  concerning  the 
causation  of  pellagra,  each  of  which  includes  a  multitude  of  hypotheses. 
According  to  one  group  of  theories,  pellagra  is  a  food  poisoning  due  to 

*  Prepared  by  W.  J.  MacNeal. 
55 


866   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

eating  maize  (Indian  corn) ;  according  to  the  other,  pellagra  is  a  specific 
infectious  disease  not  necessarily  associated  with  the  ingestion  of  corn. 
None  of  the  theories  concerning  causation  is  supported  by  conclusive 
evidence.  The  evidence  against  the  corn  theory  marshalled  by  Sambon 
and  others  has  greatly  weakened  the  almost  general  belief  in  this 
theory  which  formerly  obtained.  Some  prominent  zeists  have  recently 
shown  a  tendency  to  ascribe  pellagra  not  essentially  to  the  use  of  maize 
but  to  a  supposed  deficiency  or  lack  of  a  necessary  something  in  the 
diet.  This  change  of  opinion  has  been  caused  in  part  by  the  failure 
of  the  maize  theory  when  put  to  the  test  of  actual  observation  and  in 
part  by  an  eager  application  to  pellagra  of  the  facts  learned  in  the 
study  of  another  disease,  namely,  beriberi.  To  the  writer  it  seems 
very  improbable  that  this  new  phase  of  the  dietary  theory  will  survive 
as  long  as  has  the  maize  theory  proper,  although  it  has  received 
enthusiastic  support  from  the  U.  S.  Public  Health  Service. 

According  to  the  second  theory,  pellagra  is  a  specific  infectious  dis- 
ease, in  which  poor  nutrition  is  one  of  the  important  predisposing  factors. 
The  epidemiological  study  of  pellagra,  as  it  has  developed  and  spread 
in  certain  parts  of  the  southern  United  States,  has  brought  to  light  evi- 
dence of  its  infectious  nature  which,  to  the  writer,  seems  very  convinc- 
ing. The  same  investigations  have  also  strongly  suggested  that  the 
infection  is  intestinal  and  transmitted  in  much  the  same  way  as  is 
typhoid  fever.  A  specific  microbic  cause  of  pellagra  has  not  been 
identified. 

The  final  decision  in  regard  to  the  essential  nature  of  pellagra  must 
therefore  await  further  research.  Certain  facts  in  regard  to  the  disease 
are,  however,  well  established.  In  the  first  place  modern  students  agree 
that  the  ingestion  of  maize  is  not  essential  for  the  production  of  pella- 
gra. It  occurs  in  persons  who  have  not  eaten  this  food.  The  preval- 
ence of  pellagra,  especially  in  institutions,  bears  a  very  definite  relation 
to  the  deficiency  of  animal  protein  in  the  diet,  as  was  first  pointed  out 
in  this  country  by  the  Illinois  State  Pellagra  Commission.  This  com- 
mission observed  that  pellagra  diminished  in  the  Peoria  and  Dunning 
institutions  coincidently  with  an  increase  in  meat  supply,  while  at  the 
Elgin  Hospital  the  number  of  pellagrins  increased  with  a  decrease  in 
the  amount  of  meat  provided  per  capita.  This  commission  made  a 
specific  recommendation  to  the  Governor  of  Illinois  that  "as  a  prophy- 
lactic measure  the  animal  protein  content  of  the  State  Hospital  dietaries 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      867 

be  increased."  This  finding  of  the  Illinois  Commission  has  been 
confirmed  by  subsequent  investigators.  Thus  the  Thompson-Mc- 
Fadden  Pellagra  Commission  found,  in  1913,  that  the  individuals  in 
Southern  cotton-mill  villages,  in  whose  families  milk  was  an  article  of 
daily  use,  were  distinctly  less  subject  to  pellagra  than  their  neighbors 
who  did  not  use  milk.  More  recently  the  dietary  studies  of  the  U.  S. 
Public  Health  Service  have  confirmed  these  findings,  especially  in 
respect  to  the  value  of  milk.  However,  the  study  of  actual  dietaries 
of  pellagrins  and  their  comparison  with  dietaries  of  other  people  in  the 
same  district  has  pointed  clearly  to  the  conclusion  that  there  is  no 
single  element  in  these  diets  nor  any  group  of  elements,  the  inclusion 
or  exclusion  of  which  can  be  regarded  either  as  the  cause  or  as  a  certain 
preventive  of  pellagra. 

The  Illinois  Commission,  in  1911,  placed  as  its  first  conclusion 
"  According  to  the  weight  of  evidence  pellagra  is  a  disease  due  to  infec- 
tion with  a  living  microorganism  of  unknown  nature."  The  Thomp- 
son-McFadden  Commission  in  1913  found  that  "new  cases  of  pellagra 
originated  almost  exclusively  in  a  house  in  which  a  preexisting  pellagrin 
was  living,  or  next  door  to  such  a  house,  suggesting  that  the  disease 
has  spread  from  old  cases  as  centers."  Such  spread  was  most  rapid 
where  insanitary  methods  of  sewage  disposal  were  in  use.  In  a  later 
report,  1917,  this  commission  confirmed  these  conclusions  and  pre- 
sented the  details  of  an  extensive  experiment  conducted  in  the  com- 
munity of  Spartan  Mills,  Spartanburg,  S.  C.,  where,  by  replacing  the 
insanitary  surface  privies  with  an  efficient  water  carrier  sewer  system, 
one  of  the  worst  pellagra  foci  was  transformed  into  a  community  in 
which  the  disease  no  longer  spread.  These  observations  have,  in  all 
essentials,  been  confirmed  by  Jo*bling,  Petersen  and  their  co-workers, 
(1916,  1917)  at  Nashville,  Tenn.,  who  found  pellagra  to  be  "practically 
a  disease  of  the  unsewered  city  areas,  a  family  disease  or  almost  as 
frequently  a  disease  of  the  house  next  door." 

These  investigations  have,  therefore,  not  only  shown  that  endemic 
pellagra  may  be  alleviated  by  improvement  in  dietary  and  that  its 
further  spread  can  be  effectively  checked  by  sanitary  measures  directed 
to  the  proper  disposal  of  sewage,  but  also  have  pointed  to  the  intestinal 
tract  as  the  location  in  which  the  parasitic  cause  of  pellagra  is  to  be 
sought. 


868  MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

REFERENCES 

Sambon,  Brit.  Med.  Journ.,  Nov.  n,  1905;  Journ.  Trop.  Med.  and  Hyg.,  1910, 
Vol.  13,  pp.  271-282;  287-300;  305-315;  319-321- 

Billings  and  collaborators,  Report  of  the  Pellagra  Commission  of  the  State  of 
Illinois,  Springfield,  111.,  1912.  -A  condensation  of  this  report  appeared  in  Arch. 
Int.  Med.,  Aug.  and  Sept.,  1912,  Vol.  10. 

Sandwith,  Trans.  Soc.  Trop.  Med.  and  Hyg.,  1913,  Vol.  6,  pp.  143-148. 

Siler,  Garrison  and  MacNeal,  First  Progress  Report  of  the  Thompson-McFadden 
Pellagra  Commission,  New  York,  1913;  Second  Progress  Report  of  the  Thompson- 
McFadden  Pellagra  Commission,  New  York,  1914;  Third  Report  of  the  Robert  M. 
Thompson  Pellagra  Commission,  New  York,  1917.  Individual  papers  constituting 
the  First,  Second  and  Third  Reports  appeared  in  American  Journal  of  the  Medical 
Sciences  in  1913,  in  Archives  of  Internal  Medicine,  1914,  and  ibid.,  1916  and  1917. 

MacNeal,  The  alleged  production  of  pellagra  by  an  unbalanced  diet,  Jour.  Amer. 
Med.  Assoc.,  Mar.  25,  1916,  Vol.  66,  pp.  975-977. 

Goldberger  and  collaborators,  A  study  of  the  diet  of  nonpellagrous  and  of  pella- 
grous  households,  Journ.  Amer.  Med.  Assn.,  Sept.  21,  1918,  Vol.  71,  No.  12,  pp.  944- 
949.  Other  references  are  given  in  this  article. 

Jobling,  Peter  sen  and  collaborators,  Journ.  Infectious  Diseases,  1916,  Vol.  18, 
PP-  501-567;  ibid.,  1917,  Vol.  21,  pp.  109-131. 

RABIES* 

Lyssa  or  Rabies,  the  madness  of  dogs,  was  recognized  as  a  definite 
disease  of  animals  and  man  by  the  peoples  of  ancient  times.  The 
disease  is  generally  distributed  throughout  the  civilized  world  except 
in  those  places  where  special  measures  to  stamp  it  out  have  been 
enforced.  It  does  not  arise  spontaneously  but  is  an  infectious  disease 
transmitted  from  animal  to  animal.  Rabies  is  primarily  a  disease  of 
wolves  and  dogs,  and  the  bite  of  a  mad  dog  is  the  most  frequent  cause 
of  the  disease  in  other  animals  and  in  man.  It  is  not  uncommon  in 
horses  and  cattle,  and  all  mammals  appear  to  be  susceptible  to  it. 

In  animals  inoculated  by  injection  of  the  most  virulent  virus  (fixed 
virus)  directly  into  the  brain,  the  symptoms  of  rabies  appear  in  four 
to  six  days  and  death  usually  occurs  on  the  seventh  day.  Accidental 
inoculation  by  the  bite  of  a  rabid  animal  (street  virus)  rarely  causes  the 
symptoms  to  appear  before  three  weeks,  and  the  onset  may  be  delayed 
for  six  months  or  a  year.  Not  all  persons  or  animals  bitten  by  rabid 
animals  take  the  disease;  probably  not  more  than  one  in  four  or  five. 
This  variability  depends  upon  several  factors,  the  most  important 
ones  being  the  virulence  and  the  amount  of  disease  virus,  and  the  part 

*  Prepared  by  W.  J.  MacNeal. 


MICROBIAL  DISEASES  OF  MAN  AND  DOMESTIC  ANIMALS      869 

of  the  body  into  which  it  is  introduced.  Bites  upon  the  face  or  hands, 
because  of  the  rich  nerve  supply  of  these  regions  and  the  lack  of  protec- 
tion by  clothing,  are  likely  to  result  in  rabies  sooner  than  bites 
elsewhere. 

After  the  disease  has  developed,  death  is  inevitable.  In  all  animals 
the  symptoms  are  those  of  a  nervous  disorder.  At  first  there  is  excita- 
tion, and  this  is  followed  by  paralysis  and  death,  the  relative  length 
of  the  two  stages  varying  in  different  animals.  In  the  dog  the  disease 
runs  its  course  in  six  to  eight  days.  It  begins  with  altered  behavior  of 
the  animal,  itching  of  the  infecting  scar,  changed  appetite,  and  slight 
fever.  The  dog  swallows  grass,  stones,  and  pieces  of  wood.  As  the 
stage  of  excitement  becomes  more  fully  developed,  the  animal  may 
run  away  and  may  travel  fifty  miles  or  more,  snapping  and  biting  from 
time  to  time,  as  the  fits  seize  him,  everything  in  his  path.  Finally  the 
excitement  is  succeeded  by  paralysis,  beginning  in  the  lower  jaw,  which 
hangs  down.  Then  the  hind  legs  fail,  and  soon  the  dog,  no  longer  able 
to  drag  himself  along,  lies  completely  paralyzed,  greatly  emaciated, 
and  soon  dies.  In  the  rabbit  the  stage  of  excitement  is  hardly  notice- 
able, but  the  animal  passes  quickly  into  the  paralytic  stage,  dying  after 
two  or  three  days.  This  type  of  paralytic  rabies  sometimes  occurs  in 
dogs,  but  is  more  commonly  observed  in  herbivorous  animals. 

In  man  there  is  a  first  psychical  change,  irritation  in  the  scar  of  the 
infecting  wound  and  rise  of  temperature.  The  first  diagnostic  symp- 
tom is  usually  a  sudden  spasm  of  the  pharynx  upon  an  attempt  to 
swallow  water.  This  convulsive  seizure  is  repeated  upon  every  at- 
tempt to  drink,  and  soon  even  the  sight  of  water  or  the  thought  of  it 
brings  on  the  attack.  The  cramps  extend  to  other  muscles  of  the 
body,  and  the  patient  may  die  in  a  convulsive  seizure,  or  may  pass  into 
the  succeeding  paralytic  stage  and  die  peacefully.  The  dread  of  water 
which  is  often  so  prominent  a  symptom  in  man  has  given  the  name  of 
hydrophobia  to  the  disease.  Consciousness  and  general  intelligence 
are  not  particularly  affected.  The  duration  of  active  symptoms  of  the 
disease  is  from  three  to  six  days. 

Rabies  can  be  transmitted  with  certainty  by  injecting  a  small 
amount  of  emulsified  spinal  cord  of  the  rabid  animal  into  the  brain  of 
a  rabbit  or  guinea-pig.  Inoculation  under  the  skin  is  not  quite  so 
certain,  and  inoculation  into  the  blood  stream,  or  by  feeding,  generally 
fails  to  transmit  the  disease.  When  first  removed  from  a  rabid  dog, 


870  MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

the  virus  (street  virus)  kills  rabbits  in  from  two  to  four  weeks,  but  after 
repeated  transfer  from  rabbit  to  rabbit  in  series,  the  period  of  incuba- 
tion is  shortened  until  death  occurs  quite  regularly  in  six  or  seven  days 
after  inoculation.  Beyond  this  there  is  no  further  increase  in  virulence 
for  rabbits,  and  this  six-  or  seven-day  virus  is  called  the  "fixed  virus." 

The  localization  of  virus  in  the  body  of  the  rabid  animal  has  been 
worked  out  by  experimental  inoculations.  The  central  nervous  system 
is  always  virulent,  as  are  also  the  salivary  glands  and  the  saliva.  The 
peripheral  nerves  frequently  contain  the  virus,  less  commonly  other 
glands  and  secretions  such  as  the  tears,  urine  and  milk.  The  virus  has 
never  been  found  in  the  liver  or  spleen,  or  in  the  blood.  Under  ordinary 
conditions,  the  chief  source  of  danger  is  the  saliva  of  the  rabid  animal, 
especially  when  this  is  introduced  into  a  wound. 

Rabies  may  be  recognized  in  a  dog  in  one  of  the  three  ways :  observa- 
tion of  the  course  of  the  disease;  autopsy;  inoculation  of  test-animals 
and  observation  of  the  course  of  the  disease  in  them.  If  the  suspected 
dog  is  chained  or  caged,  the  question  of  rabies  may  be  settled  in  a  few 
days,  for,  if  mad,  the  raging  stage  will  be  succeeded  by  the  character- 
istic paralysis  and  death.  If  the  dog  has  already  been  killed,  a  careful 
autopsy  may  show  the  absence  of  normal  food  from  the  digestive 
tract  and  the  presence  there  of  abnormal  ingested  material,  highly 
suggestive  of  rabies.  Microscopic  examination  of  the  central  nervous 
system  is,  in  the  hands  of  an  expert,  a  reliable  method  of  diagnosis, 
which  in  this  case  depends  upon  the  finding  of  the  characteristic  Negri 
bodies  in  the  specimen.  For  confirmation  of  the  diagnosis,  a  portion 
of  the  brain  or  spinal  cord,  removed  without  contamination,  should  be 
injected  into  the  brain  of  test  animals,  and  the  effects  observed.  This 
last  test  carried  out  by  experienced  observers  is  justly  regarded  as  the 
most  trustworthy  of  all. 

THE  NEGRI  BODIES. — The  peculiar  bodies  found  by  Negri  in 
the  central  nervous  system  of  rabid  animals  seem  to  occur  invariably 
and  exclusively  in  this  disease,  and  it  is  probable  that  they  represent 
stages  in  the  development  of  the  infectious  agent.  These  bodies  are 
especially  numerous  and  most  easily  demonstrated  in  the  Ammon's 
horn  of  the  brain  in  cases  of  the  natural  disease  in  dogs  (street  rabies) . 
Excellent  results  may  be  obtained  by  the  method  of  Lentz.* 

•  Lentz,  Otto,  Ein  Beitrag  zur  Faerbung  der  Negrischen  Koerperchen,  Centralbl.  f.  Bakt. 
etc.,  I  Abt.,  Bd.  XLIV,  pp.  374-378. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      871 

Transverse  sections,  2  to  3  mm.  in  thickness,  of  the  Ammon's  horn  of  the  sus- 
pected brain  are  hardened  in  acetone  at  37°  for  one  hour,  then  transferred  to 
melted  paraffin  (melting  point  55°)  in  the  paraffin  oven  at  58°  for  one  and  one-half 
hours  and  embedded.  Sections,  2  to  3/x  in  thickness,  are  then  cut  with  the  micro- 
tome, floated  upon  lukewarm  water  and  mounted  upon  perfectly  clean  flamed 
glass  slides.  The  excess  of  water  is  carefully  removed  with  filter  paper  and  the 
slides  are  then  completely  dried  on  a  warm  plate  at  45°  or  in  the  incubator  at 
37°.  The  sections  adhere  perfectly  as  a  rule  and  are  dry  enough  to  proceed  with 
after  ten  to  fifteen  minutes.  The  slides  are  next  transferred  to  xylol  to  remove 
the  paraffin  and  thence  to  absolute  alcohol.  The  staining  procedure  is  as  follows: 

1.  One  minute  hi  alcoholic  eosin. 

Eosin  extra  B-Hoechst,  0.5. 
Alcohol,  60  per  cent,  100.0. 

2.  Wash  in  water. 

3.  One  minute  in  Loeffler's  methylene  blue. 

Saturated  alcoholic  solution  of  methylene  blue,  B-Patent  Hoechst,  30.0. 
Potassium  hydroxide  solution,  o.oi  per  cent  100.0. 

4.  Wash  in  water. 

5.  One  minute  in  Gram's  solution. 

Iodine,  i.o. 
Potassium  iodide,  2.0. 
Distilled  water,  300.0. 

6.  Wash  in  water. 

7.  Methylic  alcohol  until  the  preparation  becomes  entirely  red. 

8.  Wash  in  water. 

9.  Loeffler's  methylene  blue  again  for  thirty  seconds. 

10.  Wash  in  water. 

11.  Dry  carefully  by  pressing  with  filter  paper  upon  the  preparation. 

12.  Differentiate  in  alkaline  alcohol  until  only  a  weak  eosin  color  remains  in  the 
preparation. 

Absolute  alcohol,  30.0  c.c. 

Sodium  hydroxide,  i  per  cent  solution  in  absolute  alcohol,  5  drops. 

13.  Differentiate  in  acid  alcohol  until  the  collections  of  ganglion  cells  in  the 
gray  matter  are  still  faintly  blue  while  the  rest  of  thg  section  is  free  from  blue 
(macroscopic). 

Absolute  alcohol,  30.0  c.c. 
Acetic  acid,  50  per  cent,  i  drop. 

14.  Wash  quickly  in  absolute  alcohol. 

15.  Xylol. 

1 6.  Balsam  and  cover-glass. 

Steps  5  to  9,  inclusive,  may  be  omitted  to  save  time  at  some  sacrifice  In  the 
final  result.  The  Negri  body  is  stained  pink  with  blue  granules  in  its  interior. 
The  nerve  cells  are  stained  pale  blue. 


872    MICROBIOLOGY  OF  DISEASES   OF   MAN  AND  DOMESTIC  ANIMALS 

Although  sections  are  most  satisfactory  for  diagnostic  purposes  and  especially 
to  show  the  relation  of  the  Negri  bodies  to  the  ganglion  cells,  it  is  usually  possible 
to  recognize  the  Negri  bodies  in  smears,  after  a  little  experience.  For  this  purpose 
a  portion  of  the  gray  matter  of  the  Ammon's  horn  is  crushed  by  gentle  pressure 
between  two  perfectly  clean  flamed  slides  and  spread  upon  them  by  carefully  slip- 
ping the  slides  apart.  The  moist  smears  are  at  once  fixed  in  methyl  alcohol  for 
one  minute,  then  washed  in  absolute  ethyl  alcohol,  whereupon  they  are  ready  to  be 
stained  by  the  procedure  outlined  above. 


FIG.  182. — Section  through  the  cornu  ammonis  of  brain  of  a  rabi4  dog;  stained  by 
the  method  of  Lentz.  Five  Negri  bodies  of  different  sizes  are  shown,  enclosed  within 
the  ganglion  cells.  The  smallest  contains  only  three  minute  granules.  (After 
Lentz,  Centralbl.f.  Bakt.  1907,  Abt.  I,  Vol.  XLIV,  p.  378.) 

The  Negri  bodies  (Fig.  182)  appear  as  round  or  somewhat  tri- 
angular structures,  for  the  most  part  inside  the  ganglion  cells.  Their 
size  varies  considerably,  from  i/z  to  271*  in  diameter,  the  majority 
measuring  about  5/1.  In  the  interior  of  the  Negri  body,  smaller 
structures  of  variable  size  and  number  can  be  seen.  These  granules 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      873 

may  be  differentially  stained  as  in  the  Lentz  method.  Some  careful 
students  of  rabies  regard  the  Negri  bodies  as  protozoa  and  consider 
them  to  be  the  infectious  agent.  Proof  of  this  belief  is  still  lacking 
inasmuch  as  it  has  not  yet  been  conclusively  shown  that  they  are 
actually  living  organisms. 

A  wound  infected  by  a  rabid  animal  should  be  thoroughly  cauter- 
ized, under  anaesthesia  If  desired,  at  the  earliest  possible  moment,  and 
this  cauterization  should  not  be  omitted  even  if  twenty-four  hours  have 
elapsed.  Cauterization  cannot  be  relied  upon  to  prevent  the  develop- 
ment of  rabies,  but  it  does  serve  to  prolong  the  incubation  period.  The 
Pasteur  treatment  should  then  be  instituted  as  soon  as  possible,  and 
it  has  proved  to  be  practically  an  absolute  preventive,  provided  the 
incubation  period  of  the  disease  is  sufficiently  prolonged  for  the  treat- 
ment to  become  effective,  and  this  is  usually  the  case.  The  treatment 
consists  in  the  daily  subcutaneous  injections  of  altered  fixed  virus  for 
a  period  of  about  three  weeks,  and  is  most  effectively  given  at  Pasteur 
Institutes  devoted  especially  to  this  work.  Valuable  animals  as  well 
as  man  may  be  successfully  treated  in  this  way. 

The  general  prevention  of  rabies  depends  almost  solely  upon  the 
efficient  control  of  all  dogs  in  a  community.  General  muzzling,  strictly 
enforced,  is  a  certain  preventive  of  rabies,  and  in  countries  where  this  is 
done  rabies  is  practically  unknown. 

SWAMP  FEVER* 

This  is  a  comparatively  new  disease  of  horses  so  far  as  definite  infor- 
mation is  concerned,  but  is  in  reality  an  old  disease  that  has  been 
described  under  a  variety  of  names  for  many  years.  It  is  known  by 
various  names  as  infectious  anaemia,  malarial  fever,  horse  typhoid, 
"plains"  paralysis,  and  pernicious  anaemia,  and  has  been  recognized 
in  many  portions  of  the  United  States  and  Canada. 

This  disease  is  usually  of  chronic  type,  but  acute  cases  have  been 
reported.  There  is  usually  a  long  illness  extending  from  a  month  to  a 
year  or  more,  and  marked  by  periods  of  fever  and  debility,  alternating 
with  periods  of  apparent  recovery.  The  phase  of  apparent  illness  is 
characterized  by  fever,  general  weakness,  and  staggering  gait,  and  the 
disease  terminates  fatally,  as  a  rule.  Some  cases  undoubtedly  termi- 
nate as  " carriers."  The  peculiar  features  of  the  disease  are  the 

*  Prepared  by  M.  H.  Reynolds. 


874   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

alternating  periods  of  illness  and  recovery,  unthriftiness  in  spite  of 
unusually  good  appetite,  pallor  of  mucous  membranes,  dropsical 
swellings  of  the  belly  and  limbs. 

It  has  been  satisfactorily  proved  that  swamp  fever  is  caused  by 
filtrable  virus  present  in  blood  and  urine  and  which  is  quite  resistant 
to  drying,  putrefaction  and  low  temperatures. 

Under  artificial  inoculation  with  blood,  the  period  of  incubation 
varies  from  ten  to  forty  days.  The  natural  method  of  infection  is 
unknown,  but  there  are  reasons  for  believing  that  infection  does  not 
easily  occur  by  way  of  the  respiratory  or  the  digestive  organs.  The 
disease  is  apparently  not  communicated  by  simply  stabling  diseased 
animals  with  healthy  animals.  A  Japanese  commission  has  incrim- 
inated certain  biting  flies. 

Distribution  in  the  body  is  very  general,  as  shown  by  the  wide  dis- 
tribution of  characteristic  lesions,  and  as  shown  by  the  fact  that  the 
blood  is  infectious. 

The  virus  which  causes  swamp  fever  reduces  greatly  the  number  of 
red  blood  corpuscles  and  also  produces  local  haemorrhages  which  are 
most  frequently  small  and  sharply  defined.  The  reduction  of  red  blood 
cells  produces  marked  pallor,  and  there  gradually  develops  noticeable 
emaciation. 

Post-mortem  lesions  in  many  cases  are  slight.  The  haemorrhages 
may  involve  subcutaneous  and  intermuscular  tissues,  liver,  spleen, 
kidneys  and  lymph  glands  and  are  rather  common  on  the  lungs  and 
heart.  Any  of  the  abdominal  organs  may  show  the  characteristic 
haemorrhages.  The  bone  marrow  has  been  reported  in  some  cases  as 
distinctly  changed  in  color,  the  yellow  marrow  of  long  bones  becoming 
dark  red.  In  some  cases  the  liver  shows  enlargement,  degeneration 
and  necrosis. 

TYPHUS  FEVER* 

Typhus  fever  (ship  fever,  jail  fever)  has  been  known  to  exist  for 
centuries  but  until  very  recently  we  have  been  without  precise  knowl- 
edge concerning  its  cause.  Typhus  is  found  in  all  parts  of  the  world; 
it  affects  man  only  and  is  characterized  by  a  high  fever  and  an  erup- 
tion on  the  skin.  The  course  of  the  disease  is  limited  and  lasts  for 
only  about  twelve  days.  In  the  years  1909  and  1910  Nicolle,  working 

*  Prepared  by  M.  Dorset. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      875 

in  Tunis,  and  Anderson  and  Goldberger,  and  Ricketts  and  Wilder 
working  in  Mexico,  showed  that  typhus  is  communicated  from  man 
to  man  by  means  of  the  body  louse  (Pediculus  vestimenti),  and  that 
the  disease  is  not  contagious  in  the  ordinary  sense  of  the  word.  Nicolle 
states  that  after  biting  a  typhus  fever  patient  the  louse  cannot  convey 
the  infection  until  the  fourth  day  thereafter  and  that  it  loses  this  power 
after  the  seventh  day.  This  indicates  a  similarity  between  the  micro- 
organisms causing  yellow  fever,  malaria  and  typhus.  The  disease  may 
be  communicated  to  monkeys  by  subcutaneous  inoculations  of  blood 
from  a  typhus  fever  patient.  The  virus  may  be  transferred  from  one 
monkey  to  another  indefinitely.  In  monkeys  recovery  from  severe 
attack  produces  a  firm  immunity.  Plotz*  has  isolated  a  small  Gram- 
positive  bacillus  which  he  believes  to  be  the  cause  of  the  disease.  At- 
tempts to  pass  the  virus  through  filters  have  been  unsuccessful  with  the 
possible  exception  of  certain  experiments  by  Nicolle.  The  virus  is 
destroyed  by  heating  from  50  to  55°. 

YELLOW  FEVER f 

Yellow  fever  is  an  acute  infectious,  non-contagious  disease  of  man 
which  is  seen  in  tropical  and  sub-tropical  countries,  particularly  the 
West  Indies,  South  America,  and  the  west  coast  of  Africa.  The  most 
notable  symptoms  of  the  disease  are  fever,  jaundice,  and  haemorrhages 
from  the  mucous  membranes,  this  latter  resulting  in  severe  cases  in 
what  is  known  as  "Black  Vomit,"  which  consists  chiefly  of  extravasated 
blood  which  has  been  changed  to  a  brown  or  black  color  by  the  action 
of  the  gastric  juice. 

Prior  to  the  brilliant  researches  of  Walter  Reed  and  his  associates 
on  the  United  States  Army  Commission  in  the  year  1900,  it  was 
generally  believed  that  yellow  fever  was  contagious,  and  that  the 
disease  was  transmitted  directly  from  infected  to  non-infected  in- 
dividuals, and  furthermore  that  the  clothing,  bedding,  and  all  materials 
which  came  into  contact  with  the  infected  subject  were  capable  of 
transmitting  the  disease.  Reed  and  his  associates,  during  the  American 
occupation  of  Cuba,  secured  a  number  of  volunteer  subjects  to  serve 
the  Commission  in  its  studies.  This  Commission  demonstrated  posi- 
tively that  yellow  fever  was  not  transmitted  to  man  in  any  other 

*  Plotz,  Jour.  Inf.  Dis.,  July,  191 5- 
t  Prepared  by  M.  Dorset. 


876   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

way  than  by  the  bite  of  a  particular  mosquito,  Aides  (Stegomyia) 
calopus  (Meigen).  These  mosquitoes  were  allowed  to  bite  patients 
suffering  from  yellow  fever  at  different  stages  of  the  disease.  Sub- 
sequently these  same  mosquitoes  were  allowed  to  bite  healthy  men 
at  different  periods  of  time  following  their  application  to  the  infected 
individual.  It  was  proved  that  the  mosquito,  in  order  to  be  capable  of 
conveying  the  disease,  must  bite  an  infected  individual  during  the  first 
three  days  of  the  fever  and  at  least  twelve  days  must  elapse  thereafter 
before  the  mosquito  is  capable  of  transmitting  the  disease  to  a  sus- 
ceptible individual. 

Quite  recently  Noguchi*  has  isolated  from  cases  of  yellow  fever  a 
spiral  microorganism  which  he  calls  Leptospira  icter aides.  He  has 
grown  this  microorganism  in  pure  cultures  which  proved  pathogenic 
for  guinea-pigs.  Infected  guinea-pigs  develop  symptoms  and  lesions 
resembling  those  of  yellow  fever  in  man.  Leptospira  icter  aides  is  cap- 
able of  passing  through  Berkefeld  filters.  The  present  indications  are 
that  it  is  the  cause  of  yellow  fever. 

DISEASES  CAUSED  BY  PROTOZOA^ 

RHIZOPODA  (von  Seibold) 

The  amoebae  are  the  most  important  of  the  parasites  belonging  to  the  rhizopods. 
Various  species  of  amoebae  are  parasitic  in  the  intestines  of  cattle,  horses,  mice,  frogs 
and  fish  as  well  as  human  beings  and  most  of  them,  like  Entamceba  coli  of  man,  are 
harmless.  One  species,  Entamceba  histolytica,  produces  a  very  severe  dis- 
ease of  man.  Entamceba  meleagridis  is  the  cause  of  a  fatal  disease  of  turkeys 
(page  879).  Entamceba  gingivalis  (buccalis)  is  a  parasite  which  is  frequently  found 
in  a  diseased  condition  of  the  gums  characterized  by  peridental  abscesses  but  is 
also  frequent  in  apparently  healthy  mouths.  Amoebae  have  also  been  found  in 
purulent  and  serous  fluids  from  the  chest  and  abdomen  as  well  as  in  urine.  The 
parasitic  species  lack  the  contractile  vacuole  which  is  a  feature  of  the  free  living 
species  that  are  commonly  encountered  so  that  it  is  not  difficult  to  distinguish  the 
two  types. 

AMOSBIC  DYSENTERY 

Entamcsba  histolytica — Schaudinn,  1903 

Syn.:  Entamceba  tetragena — Viereck,  1907 

Distribution. — Amoebic  dysentery  occurs  most  frequently  in  tropical, 
or  sub-tropical,  countries,  but  cases  of  it  occasionally  occur  in  Great 
Britain,  in  Central  Europe,  and  in  the  United  States. 

*  Noguchi,  H.,  Etiology  of  Yellow  Fever,  Jour.  Exp.  Me4-,  Vol.  XXIX,  No.  6,  et  seq.,  1919. 
t  Diseases  arranged  genetically, 
t  Prepared  by  J.  L.  Todd. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      877 

Intestinal  amoeba;  Entamoeba  coll  Losch  (Fig.  183)  and  Entamceba  histolytica 
are  both  parasites  in  the  human  intestine.  They  measure  from  15^  to  30/11  in 
diameter  and,  when  examined  in  freshly  passed  faeces,  may  be  seen  in  active 
motion.  Their  cytoplasm  contains  a  nucleus,  vacuoles,  and  food  particles.  Both 
may  multiply  by  binary  and  by  multiple  division;  the  appearance  of  certain  of  the 
encysted  forms  in  both  species  indicates  a  process  of  autogamy.  Both  of  the 
parasitic  amoebae  of  the  human  intestine  produce  a  characteristic  number 


B 


FIG.  183. — Entamceba  coli  Losch  1875.  ^-£>  various  forms  of  motile  amoebae, 
D,  the  8-nuclear  stage;  E-G,  cysts  with  nuclear  fragments;  Ht  bursting  cyst;  /; 
young  motile  amoebae.  (After  Casagrandi  and  Barbagalh,  from  Doflein.} 


of  daughter  amoebae  in  the  course  of  their  multiplication.  E.  coli  com- 
monly divides  into  eight  small  amoebae  so  that  these  organisms  may  present 
any  number  of  nuclei  below  this  number  and  occasionally  they  contain  several 
more.  The  encysted  forms  of  this  species  also  divide  into  approximately  eight 
small  amoebae.  In  E.  histolytica  the  number  produced  as  the  result  of  division 
is  more  regular;  it  is  almost  invariably  four  in  the  division  both  of  the  motile  tropho- 
zoites  and  of  the  encysted  forms.  The  character  of  the  division  thus  furnishes  the 
most  certain  criterion  in  differentiating  the  two  species.  Multiplying  forms  are 
not  always  readily  found,  however,  and  it  is  necessary  to  take  other  characteristics 
into  consideration.  The  non-pathogenic  species  (E.  coli)  is  more  sluggish  in  its 
movements,  is  generally  larger,  dull  grayish  in  appearance,  and  has  no  sharp  differ- 
entiation into  ectoplasm  and  endoplasm.  E.  histolytica  is  active,  of  a  greenish  hue 
and  the  ectoplasm  is  well  denned  and  very  clear  in  portions  extruded  as  pseudopods. 
The  nucleus  in  the  harmless  species,  commonly  centrally  situated,  is  larger  and  shows 
a  larger  amount  of  chromatin.  The  nucleus  of  the  dysentery  amcebse  is  smaller, 


878   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

poorer  in  chromatin  and  commonly  peripherally  situated  in  the  cytoplasm.  This 
species  also  devours  red  corpuscles  in  large  numbers  but  the  absence  of  these  in 
intestinal  amoebae  is  not  sufficient  basis  for  considering  them  to  belong  to  the  harm- 
less species.  The  cysts  are  passed  in  the  faeces;  and  it  is  through  the  ingestion  of 
food  or  drink,  contaminated  by  encysted  amoebae,  that  infection  is  accomplished. 
If  unencysted  amcebae  are  swallowed,  they  are  digested  by  the  acid  juices  of  the 
stomach,  whereas  encysted  amcebae  pass  through  the  stomach  unaltered  and  become 
active  in  the  alkaline  contents  of  the  intestine.  The  dysentery  amoeba  is  pathogenic 
to  certain  lower  animals,  and  kittens  have  been  found  to  be  most  favorable  for  the 
experimental  production  of  the  disease.  Monkeys  are  also  susceptible  to  a  certain 
extent. 

Entamcsba  histolytica  may  be  present  in  an  intestine  for  months  without  marked 
symptoms  resulting.  It  may,  however,  enter  one  of  the  glands  of  Lieberkuhn  and 
pass  through  it  into  the  submucosal  layer  of  the  intestine.  Bacteria  accompany  the 
amcebae  and  they,  with  the  bacteria,  cause  an  ulcer  which  spreads  through  a  local 
destruction  of  the  submucosa,  and  undermines  the  mucosal  layer  of  the  intestine. 
In  severe  cases,  when  the  ulcers  have  spread  widely,  large  areas  of  the  mucosa 
may  be  sloughed  off.  The  amcebae  lie  at  the  edge  of  the  ulcer  and  cause  it  to  enlarge 
by  working  their  way  into  sound  tissue;  once  an  ulcer  is  started,  it  is  not  impossible 
that  Entamcsba  coli  as  well  as  the  dysentery  amcebae  may  be  found  in  it.  The  latter 
live  upon  the  red  cells  or  fragments  of  intestinal  cells.  In  chronic  cases,  the  wall 
of  the  intestine  becomes  greatly  thickened. 

Ulcers  caused  by  amcebae  are  almost  always  situated  in  the  large 
intestine;  consequently,  the  symptoms  of  amoebic  dysentery  are  those 
of  inflammation  of  that  part  of  the  body.  There  is  usually  abdominal 
pain,  accompanied  by  the  passage  of  frequent,  blood-stained  stools  with 
mucus.  The  infection  may,  however,  be  accompanied  by  no  marked 
symptoms  and  there  may  be  no  diarrhoea.  There  are  usually  developed 
more  general  symptoms,  such  as  fever  and  loss  of  flesh.  The  onset  is 
frequently  very  gradual  and  insidious  and  the  disease  runs  a  chronic 
course.  If  amoebic  dysentery  causes  death,  it  usually  does  so  by  per- 
foration of  the  bowel  with  resulting  peritonitis,  by  haemorrhage  from 
the  erosion  of  a  blood  vessel,  or  by  producing  an  abscess  of  the  liver. 
Liver  abscesses  occur  not  infrequently  in  amoebic  dysentery. 

Amoebic  dysentery  is  cured  with  difficulty  although  emetine,  a 
product  isolated  from  ipecac,  has  recently  been  found  of  great  value. 
Since  the  encysted  amcebae  are  killed  by  heat,  dysentery  can  be  avoided 
by  eating  and  drinking  only  foods  and  liquids  that  have  been  cooked. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      879 

ENTERO-HEPATITIS  OF  TURKEYS 
Entamoeba  meleagridis — Smith,  1895 

Enter o-hepatitis,  or  black-head,  of  turkeys  is  caused  by  Entamceba 
meleagridis.  The  disease  is  widespread  throughout  North  America. 
It  is  a  very  fatal  affection  and  on  many  farms  it  makes  the  raising*  of 
turkeys  a  difficult  problem.  The  disease  is  characterized  by  thick- 
ening and  ulceration  of  the  caeca,  and  by  extensive  necrosis  of  the  liver. 
Entamosba  meleagridis  is  a  small  amoeba  measuring  about  8/t  to  10^  in 
diameter.  Turkeys  probably  become  infected  with  this  parasite  by 
swallowing  its  encysted  forms;  young  turkeys  may  possibly  become 
infected  from  encysted  amoebae,  which  adhere  to  the  shells  from  which 
they  were  hatched. 

There  has  been  no  efficient  treatment  devised  for  the  disease,  since 
it  is  usually  not  noted  until  far  advanced,  but  it  can  be  avoided  through 
keeping  healthy  stock  on  land  which  has  never  been  infected  by  drop- 
pings from  infected  birds,  and  by  carefully  wiping  eggs  intended  for 
hatching  with  formalin. 

FLAGELLATA  (Cohn  emend.  Btitschli) 

The  herpetomonads  and  the  trypanosomes  are  the  most  important  of  the  para- 
sitic flagellates. 

LEISHMANIA  (Ross,  1903) 

The  three  parasites  belonging  to  this  genus  which  require  mention  are  included 
by  some  authorities  in  the  genus  Herpdomonas  but  the  differences  with  respect  to 
habit  of  life  justify  the  recognition  of  a  distinct  genus.  Herpetomonads  live  in  the 
alimentary  tract  of  various  insects,  for  example,  of  the  common  blow  fly  and  are 
extracellular  parasites.  Their  bodies  in  general  are  rigid.  Leishmania  is,  on  the 
other  hand,  an  intracellular  parasite  and  in  the  flagellated  phase  of  its  develop- 
ment its  body  is  plastic  and  bends  during  locomotion.  Three  species  are 
recognized  in  association  with  three  distinct  types  of  disease  in  man.  It  is  probable 
that  all  of  the  Leishmaniases  will  be  found,  eventually,  to  be  caused  by  a  stage  in  the 
life  cycle  of  insect-borne  herpetomonads. 

KALA  AZAR 
Leishmania  donovani — Laveran  and  Mesnil,  1903 

This  disease  occurs  in  certain  parts  of  Asia.  It  was  first  noted 
in  Assam,  Northern  India. 


880  MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

It  is  caused  by  L.  donovani  (Fig.  184).  The  parasite  is  rarely  found  in  the  blood; 
when  it  is  seen  there,  it  almost  never  occurs  free  but  is  found  in  variable  numbers 
within  phagocytic  cells.  It  is,  usually,  easily  found  by  an  examination  of  the 
juice  obtained  from  the  spleen  or  lymph  nodes  by  puncture  with  the  needle  of  a 
syringe.  The  liver  is  enlarged  and  it,  also,  contains  parasites.  As  the  organisms 
are  seen  in  preparations  of  spleen  juice,  they  are  small  ovals  measuring  about  2/i  in 
length  and  !..$/*  in  width.  They  consist  of  cytoplasm,  in  which  lie  two  chromatic 
bodies,  one  of  them  large  and  rounded,  the  other  small  and  rod-like.  This  form  of 
the  parasite  may  multiply  in  the  body  of  the  host,  by  binary  or  multiple  division  . 


•1 


1 


% 


*4     * 

%•* 


FIG.  184. — Leishmania  donovani.     Free  organisms  and  several  within  cells.     (After 

Donovan,  from  Doflein.) 

If  spleen  pulp,  or  blood,  containing  such  organisms  be  placed  on  a  suitable  culture 
medium,  they  will  develop  in  three  or  four  days,  into  herpetomonad  forms. 
The  large  nucleus  becomes  the  trophonucleus  of  the  flagellate  form,  while  the 
smaller,  rod-like  mass  becomes  the  kinetonucleus,  from  which  arises  the  flagellum. 
The  method  by  which  the  infection  is  acquired  is  unknown;  it  is  probably  by  the 
bite  of  an  insect,  perhaps  a  bedbug. 

Kala  azar  is  a  chronic  disease  characterized  by  emaciation,  by  an 
irregular  fever  and  by  considerable  enlargement  of  the  spleen.  There 
is  great  loss  in  strength  and  energy. 

Although  there  may  be  periods  of  apparent  amelioration,  the  disease 
usually  progresses  steadily,  in  spite  of  treatment,  to  a  fatal  termination. 

INFANTILE  KALA  AZAR 
Leishmania  infantum — Nicolle,  1908 

Most  authorities  recognize  the  generalized  leishmaniasis  which 
occurs  in  various  countries  bordering  on  the  Mediterranean  as  a  distinct 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      88 1 

disease.  Nevertheless,  it  is  not  certain  that  Leishmania  infantum  and 
L.  donovani  are  not  identical.  It  is  confined  almost  wholly  to  young 
children  in  whom  it  usually  runs  a  fatal  course.  This  disease  has 
been  transmitted  to  lower  animals  and  the  infection  occurs  naturally 
in  dogs,  especially  those  of  infested  districts.  Recent  investigations 
indicate  that  the  dog  furnishes  a  source  of  infection  for  human  beings 
and  that  transmission  is  affected  through  the  agency  of  a  species  of  flea. 

LOCALIZED  LEISHMANIASIS  (DELHI  BOIL) 
Leishmania  tropica — Wright,  1903 

The  localized  forms  of  leishmaniasis  occur  in  widely  distributed  localities 
throughout  the  tropics,  and  numerous  local  names  have  been  applied,  Aleppo  Boil, 
Oriental  sore,  Bagdad  Boil,  Biskra  Button,  etc.  In  South  America  likewise  a 
number  of  local  names  have  been  applied,  Espundia,  Uta,  Bubas,  Braziliana  and 
Forest  Yaws.  In  certain  forms  of  the  disease  the  mucous  membranes  are  invaded 
with  loss  of  tissue  of  the  nose  and  palate  causing  great  deformity. 

The  parasites  are  found  at  the  spreading  edge  of  the  lesions.  As  they  occur  in 
the  ulcer  they  are  oval  parasites,  almost  identical  with  those  which  are  found  in  the 
spleen  of  persons  suffering  from  kala  azar.  If  infected  material  be  placed  on  a 
culture  medium,  flagellated  forms  develop.  In  many  cases  the  organisms  are 
difficult  to  find. 

Delhi  boil  is  a  painless  ulcer,  covered  by  a  dry  scab.  It  usually 
occurs  about  the  face,  or  other  uncovered  portions  of  the  body.  If  the 
sore  be  left  untreated,  it  cures  itself  after  some  months.  In  countries 
where  it  occurs,  Delhi  boil  is  particularly  liable  to  form  at  the  site  of  a 
cut  or  abrasion.  It  is  possible  that,  in  some  cases,  the  infection  may 
be  carried  to  a  wound  by  house  flies. 

The  condition  may  be  treated  by  free  excision  although  it  runs  a 
self-limited  course.  In  places  where  it  is  endemic,  care  should  be 
taken  to  avoid  the  possibility  of  infection  by  carefully  protecting  all 
wounds,  no  matter  how  small. 

TRYPANOSOMA  (Gruby,  1843) 

Trypanosomes  are  parasitic  in  insects,  fish,  reptiles,  birds,  and  mammals  in 
all  parts  of  the  world.  Many  of  them  seem  to  be  harmless  parasites;  others  cause 
very  serious  diseases. 

Sleeping  sickness,  since  it  affects  human  beings,  is  regarded  as  the  most  im- 
portant of  the  diseases  due  to  trypanosomes. 

56 


882  MICROBIOLOGY  OF  DISEASES  OF  MAN  AND  DOMESTIC  ANIMALS 

SLEEPING  SICKNESS 

Trypanosoma  gambiense — Button,  1902 

Sleeping  sickness  is  a  disease  of  man  caused  by  Trypanosoma 
gambiense;  it  is  usually  transmitted  by  the  bites  of  Glossina  palpalis,  a 
tsetse  fly.  It  is  probably  transmitted  by  all  tsetse  flies. 

In  South  Central  Africa,  a  number  of  men  have  been  infected  with 
trypanosomes  which  differ  from  Trypanosoma  gambiense.  The  most 
important  of  them  is  Trypanosoma  rhodesiense;  the  others  are  identical 
with  trypanosomes  usually  found  in  animals.  Trypanosoma  rhodesiense 
causes  a  rapidly  fatal  disease  uninfluenced  by  any  treatment.  The 
disease  caused  by  Trypanosoma  rhodesiense  has  not  been  observed  in 
epidemic  form.  Trypanosoma  rhodesiense  is  usually  transmitted  by  a 
tsetse  fly  of  another  species,  Glossina  morsitans.  In  morphology  it 
differs  from  Trypanosoma  gambiense  in  that,  in  the  blood  of  experi- 
mental animals,  forms  occur  in  which  the  trophonucleus  is  posterior 
to  the  kinetonucleus. 

Sleeping  sickness  occurs  only  in  those  parts  of  Africa  where  tsetse 
flies  exist. 

m 


FIG.  185. — Trypanosoma  gramdosum.  n,  Tropho-nucleus£3[w,  undulating 
membrane;  c,  kinetonucleus;  /,  flagellum.  X  2000  diam.  (After  Laveran  and 
Mesnil  from  Doflein.) 

Trypanosoma  gambiense  (Fig.  186)  is  somewhat  fusiform  in  shape  and  measures 
about  17^1  to  as/*  from  the  posterior  extremity  to  the  tip  of  its  flagellum.  A  large 
tropho-nucleus  is  situated  near  the  center  of  the  trypanosome;  a  smaller,  kineto- 
nucleus lies  near  its  posterior  end.  From  this  smaller  nucleus  a  filament  arises, 
which  runs  the  whole  length  of  the  parasite  and  extends  from  its  anterior  end  as 
a  free  flagellum.  Where  the  filament  runs  along  the  body,  the  periplast  extends 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      883 

over  it  to  form  the  undulating  membrane.  The  trypanosome  moves  by  means 
of  the  undulating  membrane  and  flagellum  and  also  through  the  contraction 
of  the  myonemes  which  lie  in  the  ectoplasm.  In  the  blood,  Trypanosoma 
gambiense  multiplies  by  binary  division.  It  is  not  impossible  that  it  may 
multiply  in  other  ways,  as  do  other  trypanosomes;  for  example,  a  trypanosome 
of  frogs  loses  its  locomotory  apparatus  and  forms  a  sphere,  then  the  sphere 
divides  into  many  small  spheres,  each  of  which  becomes  a  trypanosome. 
Sometimes  Trypanosoma  gambiense  loses  its  locomotory  apparatus  and  forms 
a  sphere;  these  forms  are  found  in  the  organs  of  infected  animals.  They  are  probably 
more  resistant,  resting  forms  and  a  single  trypanosome  may  be  formed  from  some 
of  them. 


1 

FIG.  1 86. — Trypanosoma  gambiense.     (After  Minchin,  from  Doflein.) 


Trypanosomiasis  is  easily  transmitted  to  susceptible  animals  by  inoculation.  It  is 
possible  that  the  disease  may  be  transmitted  occasionally,  in  this  way,  by  the  mere 
mechanical  exchange  of  infected  material,  through  an  insect's  bite,  from  an  infected 
to  a  healthy  individual.  But,  as  a  rule,  the  disease  can  only  be  transmitted  by  the 
bites  of  Glossina  palpalis  in  which  the  organism  has  developed  (Fig.  187);  the 
fly  is  not  usually  infective  until  three  weeks  after  it  has  fed  on  an  infected  per- 
son, and  it  retains  its  infecting  power  for  some  months. 

An  incubation  period  of  at  least  ten  days  intervenes  between  the 
bite  and  the  appearance  of  symptoms,  but  this  period  may  be 
much  longer,  for  trypanosomiasis  may  manifest  itself  in  apparently 
healthy  negroes  several  years  after  they  have  left  localities  in  which 
the  disease  could  have  been  acquired.  The  disease  sometimes  causes 
death  within  three  or  four  months;  but  it  may  last  for  one  or  more 
years.  It  is  a  chronic,  wasting  affection,  characterized  by  loss  of 
strength  and  energy,  and  by  an  irregular  fever.  A  change  in  the 
mentality,  red  blotches  on  the  skin,  and  enlargement  of  the  lymphatic 
glands,  are  all  early  signs  of  the  disease.  In  the  later  stages,  headache, 


884   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 


FIG,  i&f.—Glossina  palpalis.     (After  Dojlein.) 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      885 

mania,  uncontrollable  sleep,  and  other  nervous  symptoms  may  be 
present.  Death  rarely  results  from  trypanosomiasis  alone;  the  patients 
usually  succumb  to  one  of  the  secondary  infections,  to  which  their 
reduced  condition  makes  them  especially  liable.  The  symptoms  are 
clue  to  damage  done  both  by  the  mechanical  presence  of  the  parasites 
and  to  a  trypanotoxin  produced  by  them.  The  parasites  not  only  live 
in  the  blood  and  other  fluids  of  the  body  but  are  found  in  the  tissues  of 
various  organs.  They  are  distributed  throughout  the  tissue  of  the  brain 
and  their  presence  is  associated  with  infiltration  of  the  peri-vascular 
lymph  spaces  with  large  numbers  of  lymphocytes. 

The  recognition  of  trypanosomiasis  depends  upon  the  demonstra- 
tion of  the  parasites.  They  may  be  found  in  fresh  or  stained  prepara- 
tions of  the  blood,  in  the  juice  obtained  by  aspirating  an  enlarged 
lymphatic  gland,  or  in  the  cerebrospinal  fluid.  The  examination  of  the 
blood  is  the  simplest  method  of  searching  for  trypanosomes;  the 
examination  of  gland  juice  is  the  most  efficient  one. 

The  improvement  in  the  methods  of  treating  trypanosomiasis  during 
the  past  ten  years  (1901-1911)  affords  an  excellent  example  of  the  value 
of  laboratory  work.  Before  1901  arsenic,  given  in  some  inorganic  form, 
was  the  only  drug  known  to  have  any  effect  on  trypanosomiasis.  Inor- 
ganic arsenic  drives  the  parasites  from  the  blood  and  improves  the 
patient's  condition.  Unfortunately,  the  trypanosomes  usually  reap- 
pear and,  then,  they  have  become  resistant  to  arsenic  so  that  the 
patient  succumbs  in  spite  of  repeated  doses.  Many  organic  com- 
pounds of  arsenic  were  experimented  with  in  the  hope  of  finding  an 
efficient  trypanocide  and  several  valuable  drugs  have  been  found: 
"Atoxyl"  which  is  the  sodium  salt  of  para-amido-phenyl-arsenic  acid, 
acetylated  atoxyl,  and  arsenophenylglycin,  are  all  organic  compounds 
of  arsenic.  They  are  much  more  effective  than  is  arsenic  itself. 
Similar  organic  compounds  of  antimony  and  tartar  emetic  are  as 
effective,  while  certain  aniline  dyes  have  a  distinct  trypanocidal  value. 
It  has  been  found  that  trypanosomes  may  become  resistant  to  any  one 
of  these  drugs,  and  that  drugs  may  destroy  some  stages  of  the  trypano- 
some  while  they  are  unable  to  destroy  others.  In  order  to  give  the 
parasites  no  opportunity  of  acquiring  resistance  to  any  drug,  and  in 
order  to  destroy  them  at  all  stages  of  their  development,  the  following 
general  rules  are  now  observed  in  the  treatment  of  trypanosomiasis. 
The  drugs  employed  should  be  alternated,  and  they  should  be  given 


886   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

as  early  in  the  disease  as  possible,  and  in  as  large  doses  as  possible. 
It  is  probable  that  these  principles  will  be  found  to  be  of  value  in  the 
treatment  of  other  diseases  caused  by  protozoa. 

The  prevention  of  the  disease  depends  upon  the  avoidance  of  the 
water's  edge,  where  Glossina  palpalis  exists,  and  of  the  proximity  of 
persons  or  animals  infected  by  trypanosomiasis.  The  most  usually 
successful  way  of  recognizing  infected  persons  is  by  the  discovery  of 
trypanosomes  in  the  fluid  aspirated  from  their  enlarged  lymphatic 
glands.  By  experimental  inoculation,  and  by  the  examination  of 
animals  naturally  infected,  it  has  been  shown  that  wild  and  domestic 
animals  of  many  species  may  be  infected  with  the  trypanosomes  which 
are  usually  the  cause  of  sleeping  sickness.  Some  animals  are  killed  by 
the  infection.  All  of  the  larger  animals  resist  the  infection  for  very 
considerable  periods  and  it  is  possible  that  many  of  them  are  tolerant 
to  the  infection.  This  is  especially  true  of  the  larger  ruminants. 
Therefore,  antelope,  buffalo — game — should  be  driven  away  or  de- 
stroyed in  the  neighborhood  of  human  habitation  in  order  to  remove  a 
dangerous  reservoir  of  infection. 


HUMAN  TRYPANOSOMIASIS  OF  SOUTH  AMERICA 

Trypanosoma  cruzi — Chagas,  1909 

This  disease  is  caused  by  Trypanosoma  cruzi  (Schizotrypanum  cruzi) 
and  is  transmitted  by  the  bites  of  a  reduviid  insect,  Lamus  megistus. 
It  has  been  fottfid  only  in  Brazil. 

Trypanosoma  cruzi  may  be  either  free  in  the  blood  plasma  or  lie  within  a  red 
cell.  It  multiplies,  in  the  tissue  cells  of  muscles  and  organs,  by  losing  its  locomotory 
apparatus  and  forming  Leishmania-like  bodies  which  multiply  by  repeated  divisions 
and  develop  into  new  trypanosomes.  These  young  parasites  leave  the  destroyed 
tissue  cell  where  they  were  produced  and  enter  the  blood  vessels. 

The  disease  is  a  chronic  one,  characterized  by  irregular  temperature, 
by  wasting,  oedema,  and  enlargement  of  the  spleen  and  lymphatic 
glands.  It  occurs  chiefly  in  young  children  and  is  often  fatal.  It  may 
be  prevented  by  avoiding  the  insect  which  transmits  it — the  habits  of 
Lamus  resemble  those  of  a  bed  bug. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      887 

TRYPANOSOMIASES  OF  ANIMALS 

Several  diseases,  of  great  economic  importance,  which  affect 
domestic  animals,  are  caused  by  trypanosomes.  The  following  are  the 
most  important.  Tsetse-fly  disease,  or  nagana,  of  Southern  Africa,  is 
caused  by  Trypanosoma  brucei  (Plimmer  and  Bradford)  and  it  is  trans- 
mitted by  the  tsetse  fly,  Glossina  morsitans;  it  affects  all  domestic 
animals. 

In  South  America,  mal  de  caderas,  a  disease  of  horses,  is  caused  by 
Trypanosoma  equinum  (Voges) ;  it  is  probably  transmitted  by  a  biting 
fly,  Stomoxys. 


FIG.  1 88. — Colonization  in  Trypanosoma  lewisi  (Kent).     (From  Doflein.) 

All  through  Asia,  surra,  caused  by  Trypanosoma  evansi  (Steel),  is  a 
severe  disease  of  cattle  and  equines;  it  is  probably  transmitted  by 
biting  flies. 

Trypanosoma  dimorphon  (Laveran  and  Mesnil)  and  many  other 
trypanosomes,  more  or  less  closely  allied  to  it,  cause  diseases  of 


888   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

horses,  cattle,  and  other  domestic  animals  in  many  parts  of  Africa; 
they  are  probably  all  transmitted  by  the  bites  of  flies. 

One  of  the  commonest  trypanosomes  is  Trypanosoma  lewisi  (Kent). 
It  is  usually  a  harmless  parasite  and  it  is  found  in  rats  in  all  parts  of 
the  world.  It  is  transmitted  through  the  rat  flea.  Trypanosomes 
ingested  by  fleas  develop  and  are  excreted  in  a  resting  stage  with  the 
fleas'  droppings.  Rats,  swallowing  the  infected  droppings,  or  fleas, 
become  infected.  It  is  not  transmissible  to  other  mammals. 

Dourine  or  maladie  de  coit,  is  a  serious  disease  of  equines;  it  is 
caused  by  Trypanosoma  equiperdum  (Doflein).  This  disease  was 
brought  to  North  America  by  an  imported  Percheron  stallion.  It  is 
now  endemic  in  some  of  the  western  states  and  in  part  of  southern 
Alberta,  in  Canada.  It  is  transmitted  by  coitus  and,  perhaps,  rarely 
by  the  bites  of  fleas. 

A  very  large  trypanosome,  Trypanosoma  theileri  (Bruce),  occurs  in 
cattle  in  southern  Europe  and  in  Africa.  A  similar  large  trypanosome, 
Trypanosoma  americanum,  has  been  found  in  cattle  in  the  United 
States.  These  trypanosomes  seem  to  do  no  harm  to  their  hosts. 

Although  there  are  slight  differences,  the  symptoms  are  much  the 
same  in  all  the  trypanpsomiases  of  animals,  and  they  much  resemble 
those  which  occur  in  the  diseases  produced  in  men  by  trypanosomes. 
Occasionally,  as  in  nagana,  an  animal  trypanosomiasis  may  run  an  acute 
course,  and  kill  the  host  in  two  or  three  weeks,  but  usually,  they  are 
diseases  of  long  duration,  characterized  by  irregular  fever,  cedemas  and 
progressive  loss  of  'strength,  weight,  and  energy.  Localized  areas  of 
oedema  beneath  the  skin  and  about  the  genitals  are  especially  seen  in 
dourine;  Trypanosoma  equiperdum  is  most  easily  found  by  examining 
serum  obtained  by  puncturing  these  cedemas. 

The  remaining  flagellates,  mentioned  in  the  classification  on  page 
14,  are  unimportant.  They  are  usually  parasites  of  the  urinary  or 
intestinal  tracts  and  they  may  be  associated  with  inflammation  of 
these  parts. 

SPOROZOA  (Leuckart,  1879) 

This  class  contains  many  very  important  pathogenic  parasites. 

COCCIDIA  (Leuckart) 

Coccidia  of  various  species  are  parasitic  in  the  epithelial  cells  lining 
the  intestines  of  mice,  horses,  cattle,  pigs,  goats,  and  other  animals. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      889 

In  Europe,  Eimeria  stieda  (Coccidium  cuniculi)  sometimes  causes  an 
enteritis  of  cattle;  in  East  Africa,  a  coccidium  causes  a  serious  disease 
of  cattle.  Other  coccidia  kill  many  young  pigeons,  grouse,  and 
chickens.  Coccidia  have  been  found  in  the  liver  and  intestinal  tract  of 
man. 

Coccmiosis  OF  RABBIT'S 

Eimeria  stieda — Lindemann,  1865 
Syn.:  Coccidium  cuniculi 

.  The  coccidium  causing  this  disease  is  the  best  known  of  the  coccidia 
infecting  mammals. 

This  coccidium  is  parasitic  within  the  epithelial  cells  of  the  intestine  and  within 
the  epithelium  lining  the  bile  ducts.  Adult,  asexual  forms  measure  from  20;*  to 
50M  in  diameter  and  they  produce  from  30  to  200  merozoites.  The  merozoites  infect 
other  epithelial  cells  where  they  may  again  multiply  asexually  or  they  may  develop 
into  male  and  female  forms  destined  to  multiply  sexually.  One  of  the  micro- 
gametes,  produced  by  a  microgametocyte,  fertilizes  a  macrogamete  and  an  oocyst 
is  developed.  Within  the  oocyst  a  number  of  sporoblasts  form,  which  contain  two 
spores  each.  The  oocysts  are  passed  with  the  faeces  and  if  they  are  ingested  by  a 
suitable  host  the  spores  are  set  free,  when  the  cyst  reaches  the  intestine,  and  a 
new  infection  is  commenced. 

Since  the  cells  parasitized  by  the  coccidia  are  destroyed,  it  is  evi- 
dent that  a  severe  infection  may  do  a  great  deal  of  harm  and  interfere 
with  the  functions  of  both  intestine  and  liver.  The  disease  may  be 
limited  by  making  it  impossible  for  uninfected  animals  to  come  into 
contact  with  the  droppings  of  infected  stock. 

AVIAN  COCCIDIOSIS 

Coccidium  infection  is  of  frequent  occurrence  among  birds,  and  es- 
pecially those  of  domestic  varieties,  without  causing  serious  symptoms. 
It  is  known,  however,  to  cause  severe  epidemics  in  certain  species, 
and  when  present  in  milder  form  should  be  regarded  as  antagonistic 
to  health.  Entamceba  meleagridis  the  organism  of  "  Black  head"  in 
turkeys,  from  its  peculiar  relationship  to  the  tissues,  has  been  erroneously 
regarded  as  a  form  of  coccidium. 


890  MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

ILEMOSPORIDIA  (Danilewsky  emend.  Schaudinn) 

The  most  important  parasites  of  this  order  are  those,  belonging  to 
the  Genus  Plasmodium,  which  cause  malaria  in  man.  Organisms 
similar  to  these  are  parasitic  in  the  red  blood  cells  of  apes,  bats  and 
antelopes.  Proteosoma  and  Hamoproteus  are  two  genera  parasitic 
in  the  red  blood  cells  of  birds.  It  was  the  study  of  these  avian 
parasites  which  led  to  the  discovery  of  the  way  in  which  malaria  is 
transmitted  by  the  bite  of  the  mosquito. 

PLASMODIUM  (Marchiafava  and  Celli,  1885) 

At  least  three  species  of  this  genus  are  parasitic  in  man:  Plasmodium 
vivax  (Grassi  and  Feletti) ,  the  cause  of  tertian  malaria,  Plasmodium  mal- 
aria (Laveran),  causing  quartan  malaria,  and  Plasmodium  falciparum 
(Welsh),  which  causes  aestivo-autumnal  malarial  fever. 

MALARIA 

Malaria  is  a  disease  caused  by  an  amoeboid  parasite  of  the  red  blood 
corpuscles.  It  is  transmitted  by  the  bite  of  anopheline  mosquitoes 
in  which  the  parasite  has  completed  the  sexual  cycle  of  its  development. 

It  exists  in  all  parts  of  the  tropical  and  subtropical  world  (Fig.  189). 

A  young  malarial  parasite  or  sporozoit,  derived  from  the  mosquito  enters  a  red  cell 
and  supports  itself  by  living  upon  the  cell's  substance.  The  parasite  grows,  proceeds 
to  multiply  asexually  and  divides  into  a  number  of  merozoites  which  are  set  free 
by  the  rupture  of  the  red  cell.  Those  of  the  merozoites  which  escape  ingestion  by 
the  white  cells  of  the  blood  enter  red  cells  where  they  may  again  multiply  asexually,  or 
they  may  develop  into  sexual  forms.  When  blood,  containing  malarial  parasites,  is  in- 
gested by  a  suitable  mosquito,  all  the  parasites,  except  the  adult  sexual  ones,  are  di- 
gested and  die.  Soon  after  they  are  ingested,  the  macrogametocyte  extrudes  polar 
bodies  and  becomes  a  macrogamete  and  the  microgametocyte  produces  several  micro- 
gametes,  one  of  which  enters  and  fertilizes  the  macrogamete.  Through  the  fusion  of 
macrogamete  and  microgamete  a  copula  is  formed,  which  since  it  is  motile  is  called  an 
oSkinet.  This  makes  its  way  until  it  comes  to  lie  just  beneath  the  outer  surface  of 
the  mosquito's  stomach.  There  it  develops,  as  an  oocyst,  until  it  reaches  several 
times  its  original  size.  It  divides  into  a  number  of  areas,  or  sporoblasts,  each  of 
which  subdivides  to  form  many  very  small,  hair-like  sporozoites.  When  the  oocyst 
bursts,  some  of  the  sporozoites  pass  forward  to  find  their  way  into  the  salivary 
glands  of  the  mosquito,  and,  when  it  bites,  they  are  extruded,  with  the  saliva,  into  the 


DESCRIPTION  OF  PLATE  I. 

(A  diagram  reproduced  from  Greene's  Medical  Diagnosis) 
Plasmodia  of  three  varieties.     Stained  by  Wright's  stain. 

In  this  plate  the  chromatin  of  the  parasites  is  shown  in  red  while  the 
pigment  granules  appear  as  black  dots. 

THE    QUARTAN   PARASITE    (P.  malaria) 

1-9     Asexual   multiplication.     10.    Adult  gametocyte.     n.    Normal  red 
cell.     12.  Flagellating  microgametocyte. 


THE   TERTIAN   PARASITE  (P. 

13-21.  Asexual  multiplication.      22.  Flagellating  microgametocyte. 

THE    .ESTIVO-AUTUMNAL    PARASITE    (P.    falciparum) 

23-31.  Asexual  multiplication;  25  and  26  are  doubly  infected  cells. 
32-35.  Gametocytes.      36,  37.  Flagellating  microgametocyte. 


PLATE  I. 


THL  QUARTAN  PARASITE 


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MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      89 1 


body  of  the  person  from  whom  blood  is  being  sucked.  The  entry  of  a  sporozoite 
into  a  red  cell  recommences  the  cycle  of  development  which  has  just  been 
described.  If  the  adult  sexual  parasites  are  not  taken  up  by  a  mosquito  they  die 
off  in  the  blood,  but  some  of  the  female  forms  may  live  for  years  and  then  divide 
parthenogenetically,  without  a  precedent  fertilization,  to  produce  several  young 


SCHIZOCONY   (Asexual  Generation) 
in  MAN. 


© 


Merozoites 


Macrogamete($)  ©        0 


Sporozoites. 

SPOROGONY  (Sexual  Generation) 
in  the  MOSQUITO 


Oocyst  with  Sporoblasts. 


Oocygt. 


vermicide. 


FIG.  189. — Diagram  illustrating  the  human  and  mosquito  cycles  of  existence  of 
the  malaria  parasite.     (After  Martin's  General  Pathology.') 


parasites.  It  has  been  suggested  but  never  demonstrated  that  the  sporozoites 
may  enter  eggs  lying  in  the  ovaries  of  infected  mosquitoes  and  that  mosquitoes, 
hatched  from  such  eggs,  may  inherit  the  infection  from  their  parent  and  that 
they,  also,  are  able  to  transmit  malaria. 

In  fresh  preparations  of  blood,  a  malarial  parasite  is  seen  as  a  body  of  varying 
size,  which  is  more  refractile  and  of  a  lighter  color,  than  the  red  cell  which  contains 
it.  In  its  growing  phase  it  has  distinct  amoeboid  movement  and  the  pigment 
granules  lying  in  it  are  in  active  motion.  In  preparations,  stained  by  a  modification 
of  Romanowsky's  method,  every  malarial  parasite  is  seen  to  possess  a  definite  purple 
nucleus  surrounded  by  blue-staining  cytoplasm.  Young  parasites  measure  less 


892    MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

than  a  fifth  of  the  diameter  of  a  red  cell  in  width;  adult  parasites  may  completely 
fill  the  cell  which  contains  them.  Malarial  pigment  is  the  waste  product  which 
results  from  the  digestion  of  the  haemoglobin  of  the  red  cells  by  a  malarial  parasite, 
and  consequently,  since  they  have  digested  more  haemoglobin,  the  older  parasites 
contain  more  pigment  than  do  the  younger  ones.  A  mature  asexual  parasite  finally 
segments  into  a  number  of  merozoites;  Plasmodium  vivax  forms  about  eighteen, 
Plasmodium  malarias  about  eight  merozoites.  The  adult  sexual  forms  of 
Plasmodium  falciparum  are  shaped  like  a  crescent,  and  for  that  reason  it  is  described 
by  some  as  the  type  of  a  genus  Laverania,  under  the  name  of  Laverania  malaria. 
The  three  malarial  parasites  of  man  may  be  distinguished  from  one  another  by  these 
peculiarities  as  well  as  by  other,  lesser  differences  in  themselves  and  in  the  red  cells 
which  they  parasitize. 

When  a  mature,  asexual,  malarial  parasite  bursts,  it  sets  free  young 
parasites  and  a  toxin.  Practically  all  of  the  parasites,  present  in  a 
person  suffering  from  typical  acute  malaria,  mature  and  burst  at  the 
same  time  and  the  considerable  amount  of  toxin,  set  free  in  this  way, 
produces  a  paroxysm  characterized  by  chills  and  fever.  The 
parasites  of  Plasmodium  vivax  mature  in  forty-eight  hours.  Conse- 
quently, a  person  infected  by  it  has  a  chill  when  schizogony  occurs, 
on  every  third  day,  and  the  disease  caused  by  it  is  called  a  tertian 
fever.  Plasmodium  malaria  matures  in  seventy- two  hours,  causes 
an  attack  of  ague  on  every  fourth  day  and  the  disease  produced 
is  called  quartan  fever.  Patients  infected  by  Plasmodium  falci- 
parum often  have  a  quotidian  fever  with  a  daily  rise  in  the  temp- 
erature, although  a  three  day  period  may  be  recognized  in  some 
cases.  There  are  three  stages  in  the  paroxysm:  during  the  chill,  the 
patient  feels  cold;  in  the  hot  stage  he  feels  warm — his  temperature  is 
above  normal  during  both  stages;  in  the  sweating  stage  the  temperature 
falls  to  normal  and  the  patient's  discomfort  becomes  much  less. 

The  regularly  recurring  chills  and  fever  constitute  the  only  symp- 
toms characteristic  of  malaria  and  a  regular  rise  in  temperature  on  the 
third  or  fourth  days  of  an  illness  is  strongly  suggestive  of  a  malarial 
infection.  The  type  of  disease  and  the  symptoms,  produced  by  a 
malarial  infection,  may  vary  almost  indefinitely  according  to  the  pre- 
cise way  in  which  the  host  is  harmed  by  the  infection.  Consequently, 
an  enumeration  of  the  clinical  manifestations  of  malaria  is  of  less 
importance  to  a  student  than  is  an  understanding  of  the  way  in  which 
the  malarial  parasites  harm  their  host.  The  malarial  parasites  de- 
stroy the  red  cells  and  thus  cause  an  anaemia  with  the  symp- 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      893 

toms  which  result  from  it.  Secondly,  they  produce  toxins  which 
may  cause  both  acute  and  chronic  intoxications.  The  acute 
intoxications  are  seen  in  the  elevation  of  body  temperature  and  in 
unconsciousness  in  some  pernicious  forms  of  malaria;  malarial 
neuritis  is  an  example  of  chronic  intoxication.  Lastly,  malarial  para- 
sites may  do  harm  by  blocking  the  capillaries  and  causing  the  death 
of  the  cells  which  are  cut  off  from  the  circulation;  the  symptoms  which 
result  depend  upon  the  functions  of  the  cells  which  are  destroyed. 
If  the  disease  be  long  continued,  with  a  high  temperature,  the  de- 
generative changes  which  usually  result  from  chronic  disease  and 
constant  fever  are  produced  in  the  patient. 


40 


FIG.  190. — Longitudinal  section  of  Anopheles.  A,  head;  B,  thorax;  C,  abdomen; 
i,  oesophagus;  2,  salivary  glands;  3,  dorsal  reservoir;  4,  ventral  reservoir;  5,  canal 
entering  stomach;  6,  stomach;  7,  malpighian  tubes;  8,  hind-gut;  9,  rectum;  TO,  wings; 
n,  legs.  (After  Grass  I,  from  Lang  and  Doflein.) 

The  definite  diagnosis  of  malarial  fever  depends  upon  the  demon- 
stration, in  a  patient,  of  the  malarial  parasite,  or  of  the  pigment  pro- 
duced by  it. 

Quinine  has  a  specific  action  on  the  malarial  parasite  and  is  the 
most  valuable  drug  available  for  the  treatment  of  the  disease.  It  must 
be  given  promptly  in  full  doses.  Treatment  must  be  continued  until 
all  parasites  disappear  from  the  blood. 

Malaria,  since  it  is  a  disease  which  is  caused  by  a  parasite  and 
transmitted  by  an  insect,  may  be  prevented  by  measures  directed 
either  against  the  parasite  or  against  the  transmitting  agent. 
Malaria  is  caused  by  a  Plasmodium  and  transmitted  by  the  bites  of 
mosquitoes  belonging  to  the  Anophelina.  The  disease  may  be  com- 


894  MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

bated  by  destroying  the  parasite,  in  infected  persons  with  quinine,  and 
by  isolating  such  persons  under  mosquito  nets  so  that  mosquitoes  may 
never  have  an  opportunity  of  ingesting  the  parasites  which  they  harbor 
in  their  blood.  Malaria  may  also  be  prevented  by  destroying  the 
mosquitoes  which  transmit  it.  The  most  efficient  way  of  getting  rid 
of  mosquitoes  is  to  make  it  impossible  for  them  to  breed.  The  eggs 
of  a  mosquito  are  laid  in  water,  and  water  is  absolutely  necessary  for 
the  larval  and  pupal  stages,  which  must  be  passed  through  before  the 
adult  mosquito  is  produced.  Fish  destroy  developing  mosquitoes  and 
large  sheets  of  water  are  too  rough  for  them — so  mosquitoes  must 
have,  for  breeding,  rather  small  collections  of  fresh  water  free  from 
fish.  Mosquitoes  will  soon  disappear  from  a  locality  if  all  such  col- 
lections of  water,  within  a  quarter  of  a  mile  of  it,  are  filled  up,  drained, 
or  covered  with  a  film  of  coal  oil  so  as  to  make  it  impossible  for  the 
mosquitoes  to  breed  in  them.  Those  who  live  in  a  malarious  district 
should  protect  themselves  from  mosquito  bites  by  the  careful  use  of 
mosquito-netting.  By  the  simple  observance  of  these  evident  indi- 
cations, malaria  has  already  been  banished  from  several  localities  in 
which  it  was  formerly  endemic. 

BABESIA  (Stared vici,  1893) 

This  order  is  often  called  PIROPLASMA.  It  includes  many  parasites,  which 
cause  diseases  of  considerable  economic  importance  in  horses,  cattle,  sheep,  and 
dogs.  One  of  the  best-known  species  is  Babesia  bigemina,  which  causes  Red- Water 
or  Texas  Fever  of  cattle.  The  parasites  which  are  associated  with  the  numerous 
babesiases  are  distinguished  from  one  another  by  the  host  in  which  they  are  found, 
by  slight  differences  in  their  morphology  and  by  their  inoculability  into  various 
animals. 

RED  WATER 
Babesia  bigemina — Smith  and  Kilborne,  1893 

Red  water  is  one  of  the  names  given  to  a  disease  of  cattle  which  is 
characterized  by  haemoglobinuria;  in  the  United  States  it  is  often  called 
Texas  cattle  fever.  It  is  caused  by  Babesia  boms  (bigemina)  (Fig. 
191).  The  parasite  is  transmitted  by  the  bites  of  ticks,  in  North 
America,  by  Boophilus  annulatus. 

Red  water  occurs  not  only  in  the  southern  portion  of  the  United 
States  but  almost  everywhere  in  the  tropics  and  in  many  of  the  warmer 
parts  of  the  temperate  zones. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      895 

The  parasite  is  a  pear-shaped  organism  which  usually  lies  within  a  red  cell.  It 
measures  from  2p  to  4/i  in  length  and  about  in  in  breadth.  In  fresh  preparations 
they  appear  as  refractile  bodies  possessed  of  slight  amoeboid  movement;  in  stained 
preparations  they  are  seen  to  consist  of  a  blue-staining  cytoplasm  which  contains  a 
mass  of  chromatin  at  its  broader  end.  Multiplication  is  accomplished  by  simple 
division  into  two  or  more  parts;  it  is  possible  that  schizogony  and  sporogony  may 
also  occur.  The  parasites  are  often  very  scarce  in  the  peripheral  circulation 
but  are  much  more  numerous  in  the  organs  and  particularly  in  the  spleen.  The 
disease  can  be  transmitted,  experimentally,  from  bovine  to  bovine  by  the  inocula- 
tion of  blood  which  contains  parasites;  normally,  it  is  transferred  from  animal 
to  animal  by  the  bites  of  a  tick.  The  species  of  tick  which  carries  red  water  is  not 
the  same  in  all  parts  of  the  world. 


B 


D 


H 


K 


F  G 

FIG.  19 1. — Babesia  bigemina.  Various  stages  of  development  in  red  blood  cells. 
A,  young  parasite;  B,  a  twin-form;  C-E,  a  multiple  division;  F-K,  large  pear-shaped 
forms.  (After  Doflein.) 

Ten  days  intervene  between  the  bite  of  the  infecting  tick  and  the 
first  sign  of  the  infection.  The  temperature  rises,  it  may  be,  to  106°, 
or  more,  and  it  remains  high  for  a  week.  The  animal  is  evidently  very 
ill,  it  has  no  appetite,  and  it  rapidly  loses  strength  and  weight.  Many 
red  cells  are  destroyed  and  anaemia  may  be  marked.  The  urine  is 
albuminous  and  it  is  red  because  of  the  haemoglobin  which  it  contains. 
Death  may  occur  in  very  acute  cases  as  early  as  the  second  day. 
Animals  which  recover  from  a  severe  attack  are  usually  immune  to  the 
disease.  The  immunity  is  not  an  absolute  one,  however,  for  blood 


896   MICROBIOLOGY   OF  DISEASES   OF   MAN  AND   DOMESTIC   ANIMALS 

taken  from  such  recovered  animals  is  often  infective;  the  parasite 
probably  exists  in  them  in  a  latent  form  through  the  establishment  of 
a  tolerance  on  the  part  of  the  host. 

There  is  no  specific  treatment  for  babesiasis.  Some  of  the  aniline 
drugs,  used  in  the  treatment  of  trypanosomiasis,  such  as  trypan-blue, 
are  of  some  value. 

Many  districts  are  kept  free  from  red  water  by  not  allowing  cattle 
coming  from  infected  districts  to  enter  them.  Where  it  exists,  the  dis- 
ease is  controlled  by  destroying  the  ticks  on  cattle  with  poisonous 
washes  and  by  occasionally  plowing,  or  burning  over,  the  pastures  in 
order  to  destroy  ticks  which  have  dropped  to  the  ground.  In  the 
United  States,  cattle  on  some  farms  are  kept  free  from  ticks,  and  conse- 
quently from  red  water,  by  a  manoeuvre  which  takes  advantage  of  the 
way  in  which  the  tick  transmits  the  disease.  The  adult  tick  remains 
upon  her  host  until  she  is  ready  to  deposit  her  eggs;  she  then  drops  off, 
lays  her  eggs  and  dies.  The  young  ticks,  hatched  from  these  eggs, 
attach  themselves  to  new  hosts  and  it  is  through  their  bites  that  the 
disease  is  transmitted.  Therefore,  since  the  disease  is  transmitted  by 
the  progeny  of  ticks  which  have  fed  upon  infected  mammals,  susceptible 
cattle  may  be  protected  from  the  disease  by  preventing  young  ticks 
from  reaching  them.  This  may  be  done  by  not  allowing  them  to  feed 
over  fields  where  ticks  may  have  been  dropped  until  sufficient  time, 
about  ten  months,  has  elapsed  for  all  the  ticks  and  their  progeny  to 
have  died  of  starvation. 

There  are  a  number  of  parasites  which  although  closely  related  to  the 
Babesias  are  usually  placed  in  other  genera.  Most  of  these  have  been 
shown  to  be  transmitted  by  ticks  of  various  species. 

EAST  COAST  FEVER 

Babesia  parva  (Theiler,  1903) 
Syn.:  Theilerla  parva 

This  parasite  also  is  found  in  the  red  blood  corpuscles  of  cattle. 
It  is  the  cause  of  a  disease  which  is  characterized  by  severe  anaemia. 
The  intracorpuscular  forms  vary  in  form,  some  being  slender  and  rod- 
shaped,  the  others  being  more  rounded  or  pear-shaped.  They  may  be 
arranged  to  form  a  cross,  and  this  is  not  due  to  segmentation  but  to 
fortuitous  grouping  in  heavily  infected  cells.  They  are  regarded  as 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      897 

gametocytes,  for  although  such  blood  is  infectious  for  ticks  it  will  not 
produce  infection  when  injected  into  normal  cattle.  The  multipli- 
cative or  asexual  phase  of  the  organism  is  restricted  to  certain  organs, 
especially  the  lymph  nodes,  spleen  and  bone  marrow.  The  tissue  from 
these  organs  when  injected  into  normal  cattle  produces  infection. 

OROYA  FEVER 
Bartonella  bacilliformis — Strong,  Tyzzer  and  Sellards,  1915 

A  human  disease,  characterized  by  rapidly  developing  and  severe 
anaemia  associated  with  an  irregular  fever,  occurs  in  certain  mountain 
valleys  in  Peru.  The  red  blood  corpuscles  harbor  slender  rod-shaped 
and  small  rounded  organisms  in  numbers  varying  with  the  severity 
of  the  disease.  The  rods  are  frequently  arranged  in  chains  of  two, 
three  or  even  four,  and  present  deeply  stained  granules  at  one  or  both 
extremities.  Examined  in  fresh  preparations  they  are  found  to  move 
slowly  without  marked  change  of  shape  through  the  interior  of  the 
red  cell.  Cross-forms  are  rare  and  probably  represent  fortuitous 
arrangement  rather  than  segmentation  of  the  organisms.  The  endo- 
thelium  of  the  blood-vessels  .of  the  lymph  nodes,  spleen  and  liver 
contain  organisms  in  various  stages  of  development,  small  rod-shaped 
forms  similar  to  those  of  the  red  cells  eventually  being  formed.  The 
distention  of  the  endothelial  cells  is  often  sufficient  to  occlude  many 
of  the  blood-vessels  and  in  the  lymph  follicles  of  the  large  intestine 
this  has  apparently  led  to  the  necrosis  of  the  surrounding  tissue  and 
ulceration.  The  organism  of  this  disease  is  smaller  than  that  of  East 
Coast  fever,  the  rod  forms  being  very  slender  and  nuclear  material  is 
not  so  readily  differentiated.  Its  resemblance  in  other  respects, 
together  with  the  similarity  of  its  distribution  in  the  tissues,  indicates  a 
relationship  to  this  group  of  organisms. 

The  disease  has  not  been  transmitted  to  lower  animals.  Carrion, 
a  Peruvian  student,  who  inoculated  himself  with  the  blood  of  a  patient 
suffering  from  Verruga  peruviana  died  from  a  disease  which  may  have 
been  Oroya  fever,  although  the  evidence  on  this  point  is  inconclusive. 
Oroya  fever  and  Verruga  peruviana  not  infrequently  occur  simul- 
taneously in  the  same  individual,  just  as  the  latter  disease  is  frequently 
complicated  by  malaria,  and  this  together  with  the  result  of  Carrion's 
experiment  led  many  Peruvian  physicians  to  the  erroneous  belief  that 

57 


898   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

Oroya  fever  and  Verruga  were  different  stages  or  manifestations  of  a 
single  infection.  Verruga  is,  however,  readily  transmitted  to  lower 
animals.  The  mode  of  transmission  of  Oroya  fever  has  not  been 
conclusively  determined. 

ANAPLASMOSIS 

In  a  pernicious  anaemia  of  cattle,  and  at  times  in  babesiasis,  the  red 
blood  corpuscles  contain  minute,  deeply  stained,  rounded  bodies.  These 
are  frequently  in  pairs  and  are  commonly  situated  near  the  margin 
of  the  cell  so  that  they  have  been  given  the  name  Anaplasma  marginale 
(Theiler),  by  those  who  are  convinced  of  their  parasitic  nature.  They 
have  also  been  found  in  other  domestic  animals  and  similar  structures 
are  found  normally  in  all  individuals  of  certain  species,  as  for  example, 
the  mouse.  Since  the  bodies  of  this  general  appearance  which  occur 
in  normal  animals  are  evidently  to  be  regarded  as  nuclear  material 
certain  investigators  are  inclined  to  doubt  the  parasitic  nature  of 
Anaplasma. 

SARCOSPORIDIA  (Balbiani) 

Different  species  of  this  order  are  frequent  parasites  of  all  the 
domestic  animals,  of  mice  and,  occasionally,  of  man.  Mice  are  killed 
by  them  and  it  is  possible  that  they  may  produce  ill  effects  in  men  and 
domestic  animals  but  no  definite  disease  is  associated  with  their 
presence.  Though  they  may  occur  in  any  part  of  the  body,  they  are 
most  numerous  in  certain  muscles,  such  as  those  of  the  larynx  and 
oesophagus,  which  are  near  the  alimentary  canal.  For  this  reason  it 
seems  possible  that  they  may  enter  the  bodies  of  their  hosts  with  food, 
but  our  knowledge  of  their  life  history  is  incomplete. 


HAPLOSPORIDIA  (Caullery  and  Mesnil) 

One  unimportant  parasite,  Rhino sporidium  kinealyi,  belonging  to 
this  order  is  parasitic  in  man.  It  has  been  found  in  small  tumors  of 
the  nose  and  external  ear.  A  few  cases  have  been  reported  from  Asia 
and  from  North  and  South  America.  In  the  tumors  cysts  occur 
which  are  filled  with  spores. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      899 

MYXOSPORIDIA  (Biitschli) 

There  are  many  species  in  this  group  which  are  parasitic  in  fishes 
and  certain  arthropods  but  not  in  higher  animals.  The  classification 
is  based  largely  on  the  character  of  the  spores  produced.  The  latter 
are  provided  with  a  resistant  membrane  or  shell  and  with  polar 
capsules,  each  of  which  contains  a  coiled  filament  which  when 
extruded  serves  to  anchor  the  spore.  The  spores  are  produced  con- 
tinuously within  the  protoplasm  of  the  mother  organism  which  may 
be  situated  in  any  part  of  the  body  of  the  host.  Their  presence  may, 
in  severe  infections,  cause  boil-like  lesions.  Epizootics,  killing  enor- 
mous numbers  of  fish,  are  sometimes  caused  by  these  parasites. 

MICROSPORIDIA  (Balbiani) 

Protozoa  belonging  to  this  order  do  not  produce  disease  in  man. 
They  are  the  cause  of  a  disease  of  bees,  and  they  are  of  particular  interest 
because  one  of  them,  Nosema  bombycis,  causes  Pebrine,  a  serious  disease 
affecting  silk- worms  (page  937). 

INFUSORIA  (Leddermiiller,  1763) 

Most  of  the  parasitic  infusoria  occur  in  the  alimentary  tracts  of 
their  hosts.  Harmless  infusoria  are  found  in  the  stomachs  of  many 
herbivorous  animals  and  also  in  the  large  intestine  of  the  frog. 
Balantidium  coli  is  a  common  and  apparently  innocuous  parasite  of 
the  caecum  and  large  intestine  of  the  pig,  but  it  may  cause  a  severe 
and  fatal  inflammation  of  the  large  intestine  in  man.  One  or  two  other 
infusoria  occasionally  produce  similar  symptoms  in  man.  Other 
species  of  infusoria  are  parasitic  on  fish.  Some  of  these  are  harmless, 
but  some  by  finding  their  way  into  the  gills  or  beneath  the  scales,  cause 
serious  diseases. 

BALANTIDIUM  ENTERITIS 
Balantidium  coli — Malmsten,  1857 

Balantidium  coli  is  the  most  important  of  the  infusoria  parasitic  in  man  and 
may  cause  a  form  of  dysentery. 

This  organism  measures  about  150^  in  length  and  50*1  in  breadth.  It  is  covered 
with  cilia;  its  cytoplasm  is  differentiated  to  form  oral  and  anal  areas  and  it  contains 
digestive  and  contractile  vacuoles.  It  multiplies  by  simple  transverse  division, 
either  with  or  without  a  precedent  conjugation.  It  may  encyst,  and  this  is  the 
form  in  which  the  parasite  is  transmitted  from  one  host  to  another. 


900  MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

High  enemata  of  mild  antiseptics  have  been  used  in  the  treatment 
of  this  infection. 

PARASITES  or  UNCERTAIN  POSITION 

In  Panama,  there  is  a  disease  of  man,  somewhat  resembling  one 
form  of  tubferculosis,  which  is  caused  by  a  parasite  called  Histoplasma 
capsulatum.  The  only  known  stage  of  this  parasite  greatly  resembles 
the  non-motile  form  of  Leishmania  donovani;  but  it  contains  only  one, 
not  two  masses  of  chromatin.  This  organism  was  at  first  thought  to 
be  protozoon  but  is  now  considered  to  be  a  fungus. 

The  name  Toxoplasma  has  been  given  to  a  group  of  organisms  which 
usually  inhabit  the  white  blood  cells  of  vertebrates.     They  do  no 
produce  pigment.     They  have  been  found  in  animals  and  birds  of  sev- 
eral species  in  many  parts  of  the  world.     No  parasite  of  this  genus 
has  been  found  in  man. 

CHLAMYDOZOA  (Prowazek,  1907) 

This  name  is  given  to  certain  bodies  because  their  presence  excites  the  cell 
containing  them  to  produce  a  substance  which  surrounds  them  like  a  cloak.  The 
exact  nature  of  these  bodies  is  disputed;  it  is  even  doubtful  whether  they  are  para- 
sites, or  whether  they  are  merely  the  expression  of  some  morbid  change,  produced 
in  the  cells,  by  an  unseen  virus  which  causes  the  disease.  They  have  been  found 
in  trachoma,  a  disease  of  the  eyelids  of  man,  in  hydrophobia,  in  Molluscum 
contagiosum,  a  skin  disease,  in  smallpox,  in  vaccinia,  and  in  scarlet  fever.  They 
are  mentioned  with  the  protozoa  because,  if  they  are  parasites,  they  are  probably 
more  nearly  allied  to  the  protozoa  than  to  the  bacteria.  They  are  extremely  smal 
bodies,  some  measuring  only  0.25^  in  diameter.  They  are  spherical  and  occur  within 
the  cells.  In  preparations  stained  by  Romanowsky's  method  they  are  colored  like 
chromatin. 

RICKETTSIA  (Rocha-Lima) 

Rickettsia  is  the  name  generally  applied  to  a  group  of  pleomorphic  organisms 
which  are  associated  with  typus,  Rocky  Mountain  fever  and  trench  fever.  These 
organisms  vary  in  form  from  cocci  to  long  threads  of  bacilliform  organisms.  They 
stain  with  difficulty  and  have  not  been  cultivated.  They  are  transmitted  by 
insects.  The  organisms  of  trench  fever  and  typus  are  carried  by  lice;  that  oi 
Rocky  Mountain  fever  is  carried  by  a  tick.  (Wolbach,  1919,  proved  that  the 
organisms  causing  Rocky  Mountain  Spotted  Fever,  which  he  calls  Dermacen- 
troxenus  rickettsi,  is  carried  by  Dermacentor  venustus. 

ULTRAMICROSCOPIC  VIRUSES  (See  page  119) 
SPIROCILETA  (Ehrenberg,  1833) 

Many  spirochaetes  are,  apparently,  harmless  parasites  in  shell  fish,  in  the  ali 
mentary  canals  of  various  animals  and  in  the  blood  of  fish,  birds,  and  many  mammals; 
other  spirochaetes  produce  disease  in  men  and  in  lower  animals. 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      90 1 

Several  spirochaetes  are  parasitic  in  man.  Spirochata  dentium  and  Spiro- 
chata  buccalis  are  harmless  organisms  which  are  found  in  tartar,  about  the 
teeth. 

Spirochata  vincenti  occurs  in  .great  numbers  in  a  certain  form  of  sore  throat. 
Other  spirochaetes  have  been  found  in  foul  ulcers,  and  others  have  been  found  in 
association  with  bronchitis,  urethritis  and  enteritis.  All  these  are  comparatively 
unimportant  parasites.  Spiroch&ta  recurrentis,  the  cause  of  relapsing  fever,  is  the 
most  important  of  them. 

RELAPSING  FEVER 
Spirochata  recurrentis- — Lebert,  1874 

The  relapsing  fevers  may  occur  in  any  part  of  the  world.  They 
are  caused  by  spirochaetes  and  are  transmitted  by  ticks  and  lice. 


FIG.  192. — Ornithodoros  moubata.  (Murray  from  Doflein.) 
The  disease  is  carried  by  Ornothodoros  moubata  (Fig.  192)  in  Africa 
and  wherever  this  tick  exists.  It  is  similarly  carried  in  Persia  and  else- 
where by  Argas  persicus.  Other  ticks  carry  it  in  Mexico  and  in  South 
America.  In  Europe,  Asia  and  Northern  Africa,  the  disease  is  usually 
transmitted  by  lice.  There  are  grounds  for  believing  that  it  may  also 
be  carried  by  other  biting  ectoparasites  such  as  bedbugs. 

The  spirochaetes  causing  relapsing  fevers  in  man  are  sometimes 
described  as  belonging  to  different  species  mainly  because  experimental 
animals  immune  to  infection  by  one  of  the  spirochaetes  are  susceptible 
to  infection  by  another.  This  difference  is  not  of  specific  importance 
since  two  strains  can  be  developed  from  a  single  spirochaete  neither  of 
which  'affords  immunity  against  infection  by  the  other.  Because  of 


Q02   MICROBIOLOGY  OF  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS 

the  similarity  among  these  spirochaetes  in  method  of  transmission 
and  because  of  practical  identity  in  the  symptoms  and  treatment  of 
the  diseases  which  they  produce,  it  is  better  to  describe  the  relapsing 
fevers  as  one  disease  caused  by  Spirochcrtd  recurrentis. 

Spiroch&ta  duttoni  is  a  slender  organism  measuring  from  14/1  to  i6ju  in  length;  its 
thread-like  body  lies  in  a  number  of  waves,  which  vary  greatly  in  number,  according 
to  the  way  in  which  the  preparation  is  made;  consequently,  the  number  of  waves  is 
not  a  constant  character  which  can  be  relied  upon  for  the  identification  of  these 
spirochaetes.     This  spirochaete  is  composed  of  an  outer  ectoplasmic  sheath,  and  of 
an  interior  composed  largely  of  chroma  tin;  the  sheath  extends  at  either  end  into 
flagellum-like    prolongations.      Multiplication    is    usually 
accomplished  by  transverse  binary  division,  sometimes  by 
longitudinal  division.       Sometimes,    perhaps    most    often 
toward  the  end  of  an  attack  of  fever  the  spirochaetes  coil  up 
tightly,  within  a  cystlike  matrix.      Such    encysted    forms 
may  lie  within  cells,  i.e.,  liver  cells,  and  spleen  cells;  they 
are  seen  most  frequently  in  the  liver  and  spleen,  and  they 
are  always  present  in  the  alimentary  canal  of  ticks  which 
have  ingested   spirochaete-infected  blood.     The  chromatin 
.          of  both  free  and  encysted  spirochaetes  may  be  fragmented, 

chata'duttonL (After     more  or  less  regularlv-     In  the  tick>  cysts,  containing  a 

Doflein.)  spirochaete  with  fragmented  chromatin,  burst  and  set  free 

the   granules   of  chromatin.      Some   investigators   believe 

that  each  granule  develops  into  a  spirochaete,  others  that  this  represents  a  degenera- 
tion and  destruction  of  the  parasite.  It  is  not  impossible  that  some  such  method  of 
multiplication  occurs  in  man. 

The  form  and  exact  way  in  which  the  spirochaetes  are  transmitted  is  not  com- 
pletely known.  Ticks  can  transmit  the  disease  by  their  bites.  Lice,  which  have 
become  infected  from  feeding  on  patients,  do  so  only  when  they  are  crushed  and 
their  bodies  are  rubbed  into  the  wounded  skin  by  scratching  fingers.  Similarly, 
infection  may  result  when  spirochaetes  contained  in  coxal  fluid  or  faeces,  dropped 
upon  the  skin  by  a  feeding  tick,  find  their  way  into  the  wound  made  by  the 
tick's  bite.  It  is  probable  that  a  tick,  once  infected,  never  loses  its  power  to 
transmit  the  disease;  the  infection  may  be  transmitted,  from  mother  to  daughter, 
through  at  least  three  generations  of  ticks. 

An  incubation  period  of  about  five  days  intervenes  between  infection  and  the 
appearance  of  symptoms.  The  fever  is  characteristic;  it  rises  rapidly  to,  perhaps, 
105°  and  it  remains  high  for  from  three  to  five  days.  It  then  falls  suddenly  and 
there  is  no  fever  for  from  five  days  to  two  weeks.  Then  the  temperature  rises 
again.  There  may  be  from  three  to  six  such  recurrences  of  fever  before  the 
illness  ends.  The  definite  periodicity  of  the  relapses  probably  depends  upon  some 
more  or  less  regular  developmental  change  in  the  spirochaetes  since  the  latter  are 
always  most  numerous  in  the  blood  during  the  height  of  the  fever.  The  disease 


MICROBIAL  DISEASES   OF  MAN  AND  DOMESTIC  ANIMALS      QOJ 


is  not  often  fatal  and  "606"  is  a  specific  treatment  for  it.     It  can  be  prevented 
by  avoiding  lice  and  ticks. 

TREPONEMA  (Schaudinn,  1905) 
Two  species  of  this  genus  are  very  important  parasites. 

SYPHILIS 
Treponema  pallidum — Schaudinn,  1905 

This  disease,  in  all  its  diverse  forms,  is  caused  by  Treponema  pallidum. 

The  treponema  is  an  exceedingly  slender,  thread-like  organism,  with  a  waved 
body  which  measures  from  6/*  to  14^  in  length  (Fig.  194).  It  greatly  resembles  the 
spirochaetes,  but  differs  from  them  in  having  each  end  drawn  out  to  resemble  a 


FIG.  194. — Treponema  pallidum  (in  centre)  and  the  Spirochcela  rejringens. 
(Greene's  Med.  Diagnosis) 

very  slender  flagellum.  Very  little  is  known  of  the  life  history  of  the  treponema 
except  that  it  multiplies  by  transverse  division.  It  is  transmitted  by  the  contact 
of  a  lesion,  containing  the  parasites,  with  the  broken  skin,  or  with  a  mucous  mem- 
brane of  an  uninfected  person. 

Mercury  and  potassium  iodide  were  formerly  almost  exclusively 
employed  in  treating  syphilis.  The  search  for  an  efficient  drug  for 
the  treatment  of  trypanosomiasis  has  led  to  the  discovery  of  other 


904   MICROBIOLOGY  OF   DISEASES   OF   MAN  AND  DOMESTIC  ANIMALS 

drugs  which  are  of  value  in  the  treatment  of  syphilis,  such  as  atoxyl 
(the  sodium  salt  of  para-amido-phenyl-arsenic  acid)  and  its  acetylated 
derivative,  and  of  dichlorhydrate-diamido-arseno-benzol.  The  last- 
named  drug,  "salvarsan,"  "606, "  has  proved  of  great  importance  in 
the  treatment  of  syphilis. 

YAWS  OR  FRAMBOCESIA 
Treponema  pertenue — Castellani,  1905 

This  is  a  disease  of  the  tropics  which  was  formerly  confused  with 
syphilis.  It  is  characterized  by  the  presence,  on  any  part  of  the  body, 
of  more  or  less  numerous  warty  fungoid  lesions  which  tend  to  ulcerate. 
In  this  disease  a  primary  lesion  appears  after  a  period  of  incubation  and 
this  is  followed  after  another  interval  by  a  general  eruption.  It  is  not 
a  venereal  disease  as  is  the  case  with  syphilis.  It  is  caused  by  a 
slender  spirochaete,  Treponema  pertenue,  which  is  morphologically 
identical  with  the  organism  of  syphilis.  Animals  which  have  been 
immunized  to  one  of  these  diseases  are  found  to  be  still  susceptible  to  the 
other.  The  organisms  in  yaws  are  present  in  enormous  numbers  in  the 
hypertrophied  and  swollen  epidermis  of  the  lesions  whereas  in  syphilis 
they  are  confined  to  the  deeper  tissues.  The  disease  responds  more 
favorably  even  than  syphilis  to  treatment  with  salvarsan. 

OTHER  SPIROCH^ETAL  DISEASES 

LEPTOSPIRA 

Spirochaetaform  organisms  are  the  cause  of  other  diseases.  Lepto- 
spira  icter aides  occurs  in  yellow  fever.  Leptospira  icterohamorrhagia 
is  the  cause  of  infectious  jaundice.  Spirochaetes  of  a  form  similar 
to  those  sometimes  found  in  apparently  normal  rats  and  mice  occur 
in  the  blood  of  persons  suffering  from  the  fever  which  sometimes 
follows  the  bites  of,  especially,  rats. 

Spirochaetes  cause  diseases  of  geese  in  southern  Russia  and  of  fowls  in 
Brazil  and  in  other  tropical  countries.  The  spirochaete  of  fowls,  Spiro- 
chata  gallinarum,  P.  Blanchard,  is  transmitted  by  ticks,  Argas  persicus; 
the  means  by  which  the  goose  spirochsete,  Spirochata  anserina,  Sacharoff , 
is  carried  is  not  known. 


DIVISION  IX 

% 

MICROBIAL  DISEASES  OF  INSECTS 

INTRODUCTION* 

Microbial  diseases  are  of  interest  to  the  layman  from  two  economic 
standpoints : 

I.  At    certain    stages    of    their    existence,    certain  insects  have 
an  economic  value;  for  this  reason  their  breeding  is  desirable  and  any 
plague  which  devastates  their  numbers  should  be  combated.     Pas- 
teur was  the  pioneer  in  this  line,  not  only  being  the  discoverer  of  the 
first  known  bacillary  insect  disease,  flacherie  of  the  silk-worm,  but  he 
worked  out  an  efficient  method  for  its  scientific  control.     His  work  is 
the  more  notable  since  he  was  handicapped  by  the  lack  of  suitable 
methods  of  isolation  and  study  of  the  organisms  discovered. 

II.  Certain  insects  or  their  larvae  are  at  times  veritable  plagues 
laying   waste   valuable   crops   and   causing   serious  hardships,   even 
famine  and  epidemic  disease  resulting  in  many  cases.     Not  infrequently 
these  insects  naturally  become  subject  to  microbial  enemies  which  make 
heavy  inroads  on  their  numbers,  thus  checking  the  insect  plague.     Such 
an  epizootic  occurred  among  the  white  grubs  in  Michigan  in  1912. 

The  artificial  employment  of  these  microbial  enemies  naturally 
suggests  itself  as  a  means  of  voluntary  control,  and  such  experiments 
have  been  carried  out  successfully  on  a  practical  scale.  One  of  the 
best  examples  of  this  is  seen  in  the  arrest  of  the  locust  epizootic  in  Mexico 
and  the  Argentine  Republic  by  the  use  of  cultures  of  B.  acridiorum. 

Another  thing  worthy  of  note  which  has  been  mentioned  many  times 
by  those  working  with  microbial  insect  diseases,  is  the  fact  that  these 
diseases  seem  to  be  almost  explosive  in  character;  an  epizootic  among 
insects  caused  by  a  fungus  disease  is  after  a  comparatively  short  time 
entirely  wiped  out  and  another  disease  takes  its  place;  in  many  places 
bacterial  diseases  seem  to  have  almost  entirely  supplanted  the  fungus 

*  Prepared  by  Zae  Northrup  Wyant.  except  paragraphs  on  "  Miscellaneous  Fungus  Dis- 
eases" by  C.  Thorn. 

905 


906  MICROBIAL  DISEASES   OF   INSECTS 

diseases.  This  succession  of  diseases  among  insects  takes  place  with 
such  periodicity  that  those  who  are  most  intimately  connected  with 
their  study,  can  predict  very  closely  both  the  duration  of  the  epizootic 
in  progress  and  the  time  intervening  before  the  onset  of  the  next  one. 
This  same  periodicity  takes  place  more  or  less  among  the  more  highly 
organized  animals  but  the  "explosive"  character  is  greatly  modified 
by  the  length  of  the  life  cycle.  . 

BACTERIAL   DISEASE  OF    JUNE    BEETLE  LARV.E,  Lachnosterna   spp. 
Micrococcus  nigrofaciens — Northrup* 

HISTORY  AND  DISTRIBUTION. — The  characteristics  of  this  disease 
were  noted  in  1893  by  Krassilstschik,  Russia,  but  he  did  not  consider 
it  a  disease.  It  is  common  everywhere  in  the  United  States  that 
white  grubs  of  this  and  related  species  are  found;  infected  specimens 
have  also  been  received  from  Porto  Rico. 

SYMPTOMS. — The  normal  larva  is  white,  quite  firm,  covered  with 
conspicuous  hairs;  the  head  is  brown  as  are  also  the  spiracles  or  breath- 
ing pores  along  either  side.  The  diseased  larva  has  black  shiny  spots, 
sharply  circumscribed,  located  mainly  along  the  joints  of  the  legs, 
spiracles,  and  upon  the  dorsal  or  ventral  segments  of  the  white  portion. 

Badly  diseased  larvae  are  almost  entirely  black  or  brownish  black 
in  color;  the  whole  body  often  seems  to  be  in  a  state  of  advanced  putre- 
faction, yet  the  larva  still  shows  life. 

The  progressive  destructiveness  of  the  disease  is  most  marked  in 
the  affections  of  the  legs.  In  some  cases  the  infection  begins  at  the  tip 
of  the  leg  and  as  it  progresses,  the  leg,  segment  by  segment,  blackens 
and  drops  off,  leaving  the  stumps  shiny,  black,  and  sometimes  swollen 
in  appearance;  in  other  cases  the  infection  occurs  at  one  of  the  inter- 
mediate joints  or  at  the  joint  nearest  the  body  of  the  grub,  the  leg  in 
time  loosening  and  breaking  off  at  the  point  of  infection.  Within 
certain  limits  neither  the  size  nor  the  number  of  infected  areas  seems  to 
affect  the  activity  of  the  grub.  Most  grubs  are  very  active  unless  badly 
infected  with  the  disease. 

CAUSAL  ORGANISM. — Pure  cultures  of  M.  nigrofaciens  show  micrococci  of  vary- 
ing sizes,  o.9/x  and  i.2/i  to  1.4/1  diameter  with  dividing  forms;  occur  singly,  in  pairs, 
threes  (triangular),  fours  (tetrads  or  diamond  shape),  and  clumps  of  more  or  less 

*  Northrup,  Z.  A  bacterial  disease  of  June  beetle  larva,  Lachnosterna  spp.  Tech.  Bui. 
18.  Mich.  Exp.  Sta.,  1914- 


MICROBIAL  DISEASES   OF   INSECTS  907 

regular  groupings,  the  individuals  in  a  group  arranged  in  a  honeycomb-like  order; 
chains  of  more  than  three  never  observed.  Gram-positive;  stains  well  withanilin. 
dyes. 

Growth  on  agar  abundant,  beaded,  flat,  glistening,  opaque,  pale  orange  yellow, 
butyrous  consistency,  no  odor.  Cultures  newly  isolated  are  not  pigmented  and  give 
only  a  moderate  growth.  Turbidity  in  broth,  no  ring  or  pellicle,  no  gas.  Litmus 
milk  is  slightly  reduced  and  acidified,  no  curd,  yellowish  deposit  of  bacterial  cells. 
Gelatin  is  liquefied;  dextrose,  lactose  and  saccharose  not  fermented.  Moderate 
indol  production;  nitrates  reduced. 

METHODS  OF  INFECTION. — Sterilized  soil  was  inoculated  with  a 
broth  culture  of  the  micrococcus  and  in  the  soil  were  placed  apparently 
uninfected  larvae,  which  were  incised  to  imitate  accidental  abrasion. 
.Characteristic  lesions  developed  in  two  days  at  these  points.  Under 
natural  conditions  these  larvae  bite  one  another,  especially  if  they  are 
very  numerous  in  any  one  place;  this  may  account  for  the  rapid  spread 
of  the  disease.  M .  nigrofaciens  must  be  a  common  soil  organism,  espe- 
cially where  this  disease  is  common.  Parasitic  insects  or  fungi  may 
also  aid  in  making  infection  possible. 

Excessively  wet  soil  favors  the  progress  of  the  disease.  Larvae  of 
Allorhina  nitida,  the  southern  June  beetle,  are  susceptible  to  this  infec- 
tion but  less  so  than  the  Lachnosterna  spp.  The  American  cockroach, 
Periplaneta  americana,  is  also  slightly  susceptible,  the  infection  limiting 
itself  to  the  legs. 

M .  nigrofaciens  does  not  lend  itself  readily  to  the  control  of  the 
white  grubs  on  account  of  the  limiting  environmental  conditions. 

FLACHERIE,  AN  INFECTIOUS  DISEASE  OF  SILK-WORMS 
Streptococcus  bombycis — Cohn 

HISTORY  AND  DISTRIBUTION. — Flacherie  appeared  in  the  silk  indus- 
try as  an  epidemic  at  the  end  of  the  sixteenth  century.  It  was  again 
a  serious  epidemic  about  the  year  1869  in  the  silk  nurseries  of  southern 
France.  Later  it  was  found  in  Italy  and  other  neighboring  countries 
devoted  to  sericulture.  In  1870  Pasteur  recognized  flacherie  in  silk- 
worm as  a  disease  of  the  silk- worm  distinct  from  pebrine. 

SYMPTOMS. — Diseased  worms  refuse  to  eat,  become  languid;  after 
the  fourth  molt  when  they  ordinarily  climb  up  twigs  and  branches  for 
the  purpose  of  pupating,  instead  of  spinning  their  cocoons  they  stretch 
out  and  remain  motionless  until  death,  or  they  may  fall  pendant,  hang- 


908  MICROBIAL  DISEASES    OF   INSECTS 

ing  by  their  pseudofeet.  Worms  when  dead  appear  so  very  life-like 
that  it  is  necessary  to  touch  them  in  order  to  make  sure  that  they  are 
not  living.  From  this  appearance  comes  one  of  the  names  of  this 
disease  "morts-blancs." 

After  death  they  become  soft  in  a  short  time  and  assume  a  blackish 
color  in  twenty- four  to  forty-eight  hours.  The  body  is  then  filled 
with  a  brownish  fluid  swarming  with  bacteria.  Hundreds  of  worms 
in  this  condition  show  no  polyhedral  bodies  (characteristic  of  pebrine) . 
A  glance  is  all  that  is  necessary  to  distinguish  worms  dead  of  flacherie. 

CAUSAL  ORGANISM. — In  the  silk-worms  as  well  as  in  culture  Streptococcus  bomby- 
cis  forms  short  chains  of  small  cocci,  0.89/4  in  diameter;  the  chains  are  from  5.01/1  to 
H-99AI  long;  stain  well  with  anilin  dyes  and  are  Gram-positive. 

In  gelatin  plate  cultures,  colonies  are  small,  round,  yellowish-gray,  sharply  con- 
toured, finely  granulated  interior,  gelatin  not  liquefied.  Subsurface  colonies  have 
the  same  characteristics.  Gelatin  stab  cultures  are  dull  white,  not  liquefied.  Agar 
colonies  are  small,  round  with  a  slightly  undulating  contour,  deep  brown  in  color, 
finely  granulated,  moist. 

It  is  opalescent  on  glycerin  agar  and  on  ordinary  agar  when  first  isolated.  In 
broth  at  37°  a  marked  turbidity  is  manifested  after  twelve  hours  without  flocculence; 
after  long  standing  the  broth  becomes  clear.  Potato  cultures  show  an  iridescence 
which  later  becomes  a  light  gray.  Strept.  bombycis  develops  in  milk  without  curdling 
it.  It  is  a  facultative  anaerobe;  the  temperature  optimum  is  37°  but  it  develops  well 
at  20°  also.  The  streptococcus  retains  its  vitality  for  a  long  time  in  culture.  It  is 
destroyed  at  6s°-7o°  in  fifteen  minutes. 

METHODS  OF  INFECTION. — Infection  of  the  silk-worm  takes  place  by 
means  of  food  infected  either  with  the  excrement  of  sick  individuals 
or  with  the  dust  of  infected  silk- worm  nurseries  of  the  year  preceding. 

When  silk- worms  show  all  the  symptoms  of  flacherie,  if  they  develop 
into  moths  the  eggs  laid  by  these  moths  are  always  infected.  If  any 
of  the  forms  in  which  the  silk-worm  exists  during  its  life  cycle  becomes 
infected  it  is  sure  to  die  before  the  cycle  is  completed. 

Certain  environmental  conditions  favor  the  rapid  development  of 
flacherie;  high  humidity  due  to  an  approaching  storm  or  to  keeping  the 
worms  enclosed  in  a  practically  air-tight  cage  prevents  the  transpira- 
tion which  is  so  necessary  to  the  worm  after  the  fourth  molt.  Too  many 
worms  together  often  favors  the  progress  of  the  disease. 

CONTROL. — Pasteur  instituted  the  following  means  of  producing 
healthy  strains  of  the  silk- worm;  a  small  portion  of  the  digestive  cavity 
of  a  moth  was  abstracted  with  a  scalpel,  mixed  with  a  little  water  and 


MICROBIAL  DISEASES   OF   INSECTS  909 

examined  microscopically.  If  the  moths  did  not  contain  the  character- 
istic microorganism,  the  strain  they  came  from  might  unhesitatingly 
be  considered  as  suitable  for  seeding.  The  flacherie  organism  was  as 
easily  recognized  as  the  pebrine  corpuscle,  but  the  infection  was  more 
difficult  to  prevent  on  account  of  the  environmental  conditions  above 
mentioned. 

Silkworms  have  been  fed  on  mulberry  leaves  washed  with  water  or 
an  aqueous  solution  of  lysoform,  but  a  few  sporadic  cases  of  flacherie 
and  emaciation  occurred  nevertheless.* 

Phototaxy  has  been  employed  successfully  in  selecting  larvae  of 
Bombyx  mori  most  resistant  to  flacherie.  Newly  hatched  larvae 
immediately  turn  to  the  source  of  light,  while  this  movement  diminishes 
during  the  following  days  and  disappears  entirely  at  the  end  of  the 
first  stage.  During  the  subsequent  stages  there  is  an  inverse  but  less 
energetic  movement  and  the  larvae  tend  to  avoid  the  light.  The  larvae 
which  are  most  resistant  to  flacherie  are  those  which  from  the  time  of 
their  birth  had  travelled  farthest,  f 

THE  " JAPANESE  GIPSY-MOTH  DISEASE" 
Streptococcus  disparis  n.sp. — Glaser { 

HISTORY  AND  DISTRIBUTION. — During  the  summer  of  1915  a  large 
series  of  eggs  of  the  Japanese  gipsy  moth  (Porthetria  dispar  L.)  were 
hatched,  which  had  been  obtained  for  Glaser  from  Ogi,  Japan.  On 
reaching  the  third  stage  many  of  the  caterpillars  began  to  die  of  a 
peculiar  disease  which  Glaser  had  never  in  previous  years  noticed  in 
any  of  his  American  cultures.  The  infection  later  spread  to  the  Ameri- 
can race,  and  the  most  vigorous  methods  of  isolation  and  disinfection 
had  to  be  inaugurated  in  order  to  save  most  of  the  cultures  from  extinc- 
tion. As  the  disease  was  very  soon  controlled,  distinct  in  this  respect 
from  wilt  (polyhedral  disease),  a  bacterial  origin  was  at  once  suspected. 
This  disease  was  then  studied  in  the  belief  that  it  might  be  used  in 

*Sacchi,  Rosa.  Partial  disinfection  of  mulberry  leaves  in  feeding  silkworms. 
E.S.R.  39,  pp.  560-561,  1918. 

fAcqua,  C.  The  use  of  phototaxy  in  selecting  from  the  moment  of  their  birth 
those  larvae  of  Bombyx  mori  most  resistant  to  the  disease  flacherie.  E.S.R.  38, 
p.  860,  1918. 

JGlaser,  R.  W.  Anew  bacterial  disease  of  gipsy-moth  caterpillars.  Jour. 
Agr.  Res.  13,  1918,  pp.  515-522. 


9 10  MICROBIAL   DISEASES    OF   INSECTS 

combating  the  gipsy  moth  in  the  field.     Many  observations  and  tests 
show  that  this  disease  did  not  occur  in  this  country  prior  to  1917. 

SYMPTOMS. — When  a  caterpillar  contracts  this  new  disease  it  ac- 
quires a  violent  form  of  diarrhoea,  loses  its  appetite,  and  finally  ceases 
to  eat.  The  insect  seems  to  lose  all  muscular  coordination  and  usually 
crawls  to  some  elevated  place,  where  it  soon  dies.  After  death  it 
hangs  in  a  flaccid  manner  by  its  prolegs,  with  an  appearance  of  death 
from  wilt.  In  contradistinction  to  wilt,  however,  the  skin  does  not 
rupture,  but  is  so  tough  that  one  can  pick  up  and  stretch  the  animal 
with  considerable  force  before  the  skin  breaks. 

CAUSAL  ORGANISM. — A  microscopic  examination  of  smears  from  the  dead  cater- 
pillars readily  precludes  the  possibility  of  wilt.  Instead  of  polyhedra,  large  numbers 
of  a  streptococcus,  Streptococcus  disparis  are  present. 

Strept.  disparis  has  a  diameter  of  less  than  i/*;  chains  of  3  to  4^  frequent  in  liquid 
media;  division  in  one  plane;  capsulated;  non-motile;  Gram-positive;  stains 
readily. 

Cultural  characteristics  are  as  follows:  On  nutrient  agar  slant,  neutral,  growth  in 
five  days  at  35°  scanty,  beaded,  flat,  glistening,  smooth,  white,  opaque,  no  odor, 
butyrous,  medium  unchanged.  On  potato  agar  slant,  neutral,  growth  in  five  days 
at  35°  abundant,  spreading,  flat,  glistening,  smooth,  white,  opaque,  odor  absent, 
butyrous,  medium  unchanged.  Potato,  growth  moderate,  spreading,  flat,  no  odor, 
butyrous,  color  of  medium  unchanged.  Gelatin  stab,  growth  best  at  top,  beaded, 
no  liquefaction,  medium  unchanged.  Nutrient  broth,  no  ring  or  pellicle,  slight  cloud- 
ing, clearing  after  fifteen  days,  slight  sediment,  no  odor.  Milk,  coagulation  de- 
layed, extrusion  of  whey,  color  unchanged,  no  peptonization.  Litmus  milk,  acid, 
prompt  reduction,  coagulation  delayed,  extrusion  of  whey,  no  peptonization.  Dun- 
ham's peptone  solution,  clouding  very  slight,  growth  poor.  Gelatin  colonies,  growth 
slow,  colonies  very  small  and  majority  under  surface,  surface  colonies  round,  slightly 
convex,  edge  entire,  no  liquefaction.  Nutrient  agar  colonies,  growth  slow,  majority 
of  colonies  under  surface  and  oblong,  surface  colonies  round,  smooth,  convex,  edge 
entire,  internal  structure  finely  granular,  diameter  0.25  to  0.33  mm.  Potato  agar 
colonies,  growth  rapid,  majority  of  colonies  under  surface  and  oblong,  surface 
colonies  round,  smooth,  convex,  edge  entire,  internal  structure  finely  granular, 
diameter  i  to  1.5  mm.  No  ammonia  production.  Nitrates  not  reduced.  Indol 
and  H2S  not  produced.  Acid  but  no  gas  is  formed  in  the  following  carbohydrate 
broths  used,  extrose,  levulose,  saccharose,  maltose,  lactose,  mannit,  adonit,  dulcit. 
Facultative  anaerobe.  Best  media  for  cultivation  1.5  per  cent  neutral  potato  agar, 
and  neutral  nutrient  bouillon  containing  i  per  cent  of  carbohydrate,  especially 
saccharose,  maltose  or  mannit.  Pathogenic  to  caterpillars  of  the  American,  Euro- 
pean and  Japanese  races  of  the  gipsy  moth  (Porthetria  dispar  L.}.  Not  pathogenic 
to  silkworms  (Bombyx  mori  L.)  and  army  worms  (Cirphis  unipuncta  Ha  worth)  when 
fed  per  os.  Guinea  pigs,  rabbits  and  human  beings  when  fed  pure  cultures  per  os 


MICROBIAL  DISEASES   OF   INSECTS  91 1 

not  affected.  This  organism  is  distinct  culturally  and  biochemically  from  the 
organism,  Diplococcus  lymantria  recently  described  by  Paillot  which  is  also  parasitic 
in  the  gipsy  moth  caterpillar,  and  moreover  is  highly  pathogenic  to  the  caterpillars 
while  D.  lymantrice  is  not  very  pathogenic. 

METHODS  OF  INFECTION. — During  the  earlier  stages  of  the  disease 
when  the  caterpillars  contract  diarrhoea,  the  semiliquid  faeces  every- 
where soil  the  food  plants.  This  fecal  matter  is  grossly  contaminated 
with  the  streptococcus,  and  is  the  principal  cause  for  the  rapid  spread 
of  the  infection. 

PATHOLOGY.— Sections  demonstrate  that  this  bacterium,  during  the 
early  stages  of  the  disease,  is  found  throughout  the  alimentary  tract. 
Later,  and  especially  after  death,  the  intestinal  epithelium  disinte- 
grates and  ruptures,  liberating  the  organisms  into  the  body  cavity 
where  they  invade  practically  all  the  tissues. 

Microscopically  striking  changes  can  be  noted  in  the  muscle  tissues 
even  in  the  early  stages  of  the  disease.  Normal  muscle  tissue  shows 
clearly  the  striae  but  in  the  early  stages  of  the  disease  these  show  less 
clearly  and  the  individual  fibrillae  seem  to  be  loosely  arranged.  Later 
stages  of  the  disease  show  first  an  absence  of  the  typical  striated  ap- 
pearance due  to  the  fact  that  the  fibrillae  have  lost  their  compactness 
and  have  separated  from  one  another  like  threads  of  cotton;  the  arco- 
lemma  disintegrates  gradually  with  the  rest,  and  the  nuclei  of  the  cells 
lose  their  normal  positions  and  become  scattered.  Up  to  this  time  it 
can  be  safely  predicted  that  Strept.  disparis  will  be  found  in  the  ali- 
mentary tract.  Then  finally,  the  muscle  tissue  disintegrates  com- 
pletely, the  fibrillae,  etc.,  are  no  longer  visible,  and  the  whole  simulates 
coagulated  protein  material  with  minute  granules  scattered  throughout. 
When  this  stage  in  muscular  disintegration  has  arrived,  nearly  all  of 
the  other  tissues  have  likewise  disintegrated  more  or  less,  and  Strept. 
disparis  may  now  be  seen  scattered  everywhere. 

Field  experiments  were  conducted  with  Strept.  disparis  in  sections 
of  the  gipsy-moth  infested  territory  many  times  with  success.  In  two 
places  quite  a  severe  epidemic  was  created.  A  large  amount  of  work 
still  is  needed,  however,  to  determine  the  relative  importance  of  this 
method  of  combating  the  gipsy-moth. 


912  MICROBIAL  DISEASES  OF  INSECTS 

BACTERIAL  DISEASE  OF  LOCUSTS 
Bacillus  acridiorum — d'Herelle 

HISTORY  AND  DISTRIBUTION. — Tropical  and  subtropical  countries 
covering  more  than  half  the  earth's  surface  suffer  periodically  from 
plagues  of  locusts  of  different  species.  Famine  and  its  attendant,  epi- 
demic disease,  follow  in  their  wake  and  decimate  the  regions  invaded. 
A  bacterial  epizootic  has  become  a  natural  means  of  control. 

Bacillus  acridiorum,  the  cause  of  the  locust  epizootic,  was  discovered 
in  Mexico  in  the  state  of  Yucatan  by  F.  d'Herelle.  In  1909  a  certain 
mortality  was  noted  among  the  swarms  which  arrived  from  the  south 
of  the  state  where  they  winter;  the  following  year  the  epizootic  was 
generalized  and  raged  among  a  large  number  of  bands;  finally  in  1911, 
all  of  the  swarms  which  appeared  were  attacked,  and  in  1912,  the  locust 
invasion  ceased.  These  particular  locusts  were  the  Schistocerca 
pollens. 

SYMPTOMS. — The  locusts  which  are  attacked  by  the  natural  disease, 
present  symptoms  which  are  identical  with  those  which  are  experimen- 
tally inoculated  or  contaminated  per  os.  After  a  time  of  incubation, 
which  varies  from  one  to  forty-eight  hours  according  to  the  virulence 
of  the  bacillus,  the  age  and  individual  resistance  of  the  insect,  and  the 
environment  (temperature  especially),  at  first  the  contents  of  the  chyli- 
fic  stomach  become  liquefied  and  assume  a  dark  color  resembling 
coagulated  blood.  The  locust  ceases  to  eat,  becomes  flabby,  jumps 
awkwardly  and  hides  itself  under  tufts  of  herbage.  The  intestinal  con- 
tents next  become  liquefied;  they  are  at  first  a  clear  yellow,  later  dark- 
ening little  by  little  until  they  are  blackish  in  color.  At  this  stage  a 
slight  pressure  upon  the  abdomen  causes  the  liquefied  intestinal  con- 
tents to  issue  from  the  anus  and  the  characteristic  diarrhoea  reveals  itself 
on  the  vegetation  which  is  fouled  with  the  dejecta  of  the  sick  locusts. 
Some  hours  afterward  the  locust  falls  upon  its  side  and  the  legs  move 
spasmodically;  the  locust  remains  in  this  comatose  state  several  min- 
utes to  "several  hours  until  death  occurs.  When  the  virulence  of  the 
coccobacillus  is  very  high,  very  often  the  chylific  stomach  only  presents 
the  characteristic  blackening;  death  occurs  before  the  intestinal  con- 
tents have  undergone  a  modification.  After  death  the  insect  putrefies 
very  rapidly  and  the  tegument  becomes  dark. 


MICROBIAL  DISEASES   OF   INSECTS  913 

The  intestinal  content  of  locusts  attacked  with  this  disease  shows 
microscopically  practically  a  pure  culture  of  this  bacillus.  The  intes- 
tinal contents  of  healthy  locusts  are  poor  in  bacteria,  sometimes  seem- 
ingly aseptic.  Among  the  saprophytes  found,  the  most  common  is  a 
motile,  Gram-positive  coccobacillus  which  causes  death  of  locusts  by 
injection  but  never  by  ingestion.  It  is  distinguishable  from  the  specific 
coccobacillus  by  the  disagreeable  odor  which  it  produces  in  the  locusts 
or  in  culture  media.  Sometimes  staphylococci  are  found,  rarely  B. 
subtilis;  only  one  saprophyte  per  hundred  of  the  specific  bacillus  ren- 
ders the  isolation  of  the  latter  very  simple. 

All  of  the  tissues  of  the  locust  are  invaded  by  this  bacillus  as  has 
been  proved  microscopically.  A  pure  culture  can  be  obtained  from  the 
blood  at  the  same  time  that  the  intestinal  contents  are  attacked,  thus 
B.  acridiorum  produces  a  veritable  septicemia. 

CAUSAL  ORGANISM. — B.  acridiorum,  the  causal  organism  of  the  Mexican  locust 
epizootic  is  a  short,  slightly  ovoid  bacillus,  decidedly  polymorphous;  in  the  same 
culture  coccus,  forms  of  about  0.6/4  are  found  beside  of  forms  plainly  bacilli,  0.4/11  to 
o.6ju  by  o.gn  to  i.5ju;  actively  motile  possessing  peritrichic  flagella;  Gram-negative 
but  stains  readily  with  anilin  dyes;  the  bacillus  takes  the  stain  most  deeply  at  the 
poles,  especially  if  Ziehl's  carbol-fuchsin  is  used  for  one  to  two  seconds. 

Facultative  anaerobe;  cultures  grow  readily  from  16°  to  43°  in  all  ordinary  media, 
even  in  Raulin's  medium.  It  develops  very  rapidly  at  37°  in  broth,  turbidity  appear- 
ing at  about  the  fourth  hour.  A  delicate  membrane  is  formed  on  broth  which  clears 
only  after  three  weeks,  leaving  a  heavy  sediment.  Young  agar  colonies  are  cir- 
cular and  have  a  waxy  appearance;  they  grow  rapidly,  being  plainly  visible  after 
twelve  hours;  after  eighteen  hours,  they  are  2-3  mm.  in  diameter.  Subsurface 
colonies  are  small,  spherical,  whitish  and  opaque.  Gelatin  is  not  liquefied. 
Milk  is  coagulated  and  rendered  strongly  alkaline.  Grows  abundantly  on  potato 
having  a  creamy  appearance;  the  culture  in  the  water  at  the  bottom  of  the  tube  is 
so  dense  that  the  liquid  becomes  sirupy  and  has  a  strong  alkaline  reaction. 
Dextrose,  levulose,  maltose  and  galactose  are  fermented;  the  inoculated  medium 
containing  one  of  these  sugars  becomes  acid  at  first,  then  alkaline.  This  alkalinity 
is  due  to  the  formation  of  ammonia.  B.  acridiorum  has  lived  over  two  years  in 
sealed  tubes. 

METHODS  OF  INFECTION. — Natural. — There  are  several  natural 
methods  by  which  the  epizootic  is  spread.  Sick  locusts  or  nymphs 
leave  their  infectious  liquid  dejecta  on  the  vegetation,  the  other  locusts 
eat  the  contaminated  herbage,  contract  the  disease  and  in  turn  infect 
new  plants  thus  continuing  the  cycle.  With  certain  species  of  locusts, 
the  Schistocerca  for  example,  another  very  important  mode  of  contagion 

58 


QI4  MICROBIAL  DISEASES    OF   INSECTS 

exists:  when  one  of  their  number  becomes  weak  or  where  vegetation  is 
scarce  both  the  nymphs  and  the  adults  eat  one  another. 

At  the  time  of  depositing  the  eggs,  if  the  female  or  even  the  male 
is  diseased,  the  eggs  will  be  forcibly  soiled  with  the  liquid  of  the  diar- 
rhoea and  the  bacillus  will  be  conserved  up  to  the  time  of  hatching  upon 
the  eggs  or  in  the  mucilaginous  matter  surrounding  the  eggs  which  the 
locust  has  provided  for  their  protection. 

A  certain  number  of  locusts  among  every  swarm  act  as  healthy 
"carriers."  Carriers  among  nymphs  have  never  been  found. 

The  period  of  life  of  the  insect  affects  its  resistance.  The  adult 
locust  is  individually  much  more  susceptible  than  the  nymph.  The 
habits  of  life  of  each,  however,  have  a  great  influence.  The  nymphs 
are  continually  in  contact  with  the  vegetation  and  with  each  other  as 
they  march  in  very  dense  columns;  they  are  endowed  with  a  voracious 
appetite  and  undergo  in  the  short  period  of  their  larval  life,  five  molts, 
which  are  the  periods  of  their  least  resistance.  The  winged  locusts, 
to  the  contrary,  passing  a  large  part  of  their  life  in  the  air,  are  only 
rarely  pressed  one  against  the  other,  except,  for  example,  when  the 
weather  is  cold;  they  also  eat  much  less  than  the  nymph,  thus  the 
epizootic  will  have  a  greater  tendency  to  become  generalized  among 
the  bands  of  nymphs  than  among  the  swarms  of  adult  locusts. 

The  age  of  the  nymph  or  locust  influences  its  resistance;  the  young 
nymphs  have  a  maximum  resistance,  but  this  decreases  gradually, 
reaching  its  minimum  at  the  time  of  the  last  molt;  the  adult  locust  has 
its  minimum  of  resistance  at  the  egg-laying  period. 

The  period  of  the  molt  is  not  a  means  of  protection  against  this 
type  of  disease,  which  is  a  generalized  septicemia. 

ARTIFICIAL. — The  virulence  of  B.  acridiorum  decreases  very  rapidly  in  culture 
and  in  order  to  obtain  the  desired  destruction  of  locusts  it  is  absolutely  necessary 
to  employ  cultures  of  the  highest  possible  virulence  as  an  attenuated  virus  immunizes 
the  locust  and  renders  it  refractory  to  a  culture  of  the  highest  virulence  when  applied 
later. 

The  virulence  is  increased  by  successive  passages  through  locusts  or  nymphs; 
twelve  series  of  passages  are  made  using  twelve  locusts  in  each  series.  The  cul- 
ture to  be  rejuvenated  is  mixed  with  a  few  cubic  centimeters  of  sterile  water  or  broth. 
Injections  are  made  with  a  syringe  having  a  very  fine  sharp-pointed  needle.  The 
insect  is  seized  with  the  left  hand,  the  ventral  portion  toward  the  operatoi,  and  the 
needle  of  the  syringe  inserted  between  the  second  and  third  anterior  abdominal 
segments  at  the  point  of  intersection  with  one  of  the  longitudinal  ridges,  horizon- 


MICROBIAL  DISEASES   OF   INSECTS  915 

tally  in  the  direction  of  the  head  to  a  depth  of  about  3  mm.  for  an  adult  insect,  a 
little  less  for  a  nymph.  The  point  of  the  needle  should  enter  the  abdominal  cavity, 
not  merely  pierce  the  tegument  as  in  the  latter  case  the  effect  would  be  nil.  If 
the  needle  is  inserted  too  deep  the  internal  organs  will  be  injured.  A  very  fine- 
pointed  bent  pipette  could  be  employed  equally  well.  One  or  two  drops  of  the  emul- 
sion of  the  old  culture  are  injected. 

As  soon  as  the  locusts  in  the  first  series  become  sick  or  preferably  are  nearly 
dead,  press  the  abdomen  between  the  fingers  and  collect  in  a  watch  glass  the  blackish 
liquid  which  issues  from  the  anus.  Inject  a  drop  of  this  liquid  into  the  abdominal 
cavity  of  the  second  series  of  locusts,  following  the  same  technic  and  observing  the 
same  precautions  as  for  those  of  the  first  series.  These  insects  will  die  in  a  shorter 
period  of  time.  Obtain  as  previously,  in  a  watch  glass  the  intestinal  liquid  of  three 
or  four  of  the  first  dead  locusts  of  the  second  series,  dilute  half  with  water  and  sterile 
broth  and  inoculate  the  third  series.  To  inoculate  the  fourth  series,  use  the  intes- 
tinal liquid  of  the  first  dead  of  the  third  series  diluted  to  a  third;  a  fifth  series  with 
the  liquid  diluted  to  a  fourth  and  continue  with  the  series  in  this  way.  It  is  excep- 
tional that  it  will  be  necessary  to  proceed  further  than  the  twelve  series.  The  viru- 
lence of  B.  acridiorum  is  increased  sufficiently  if  death  occurs  eight  hours  after  injec- 
tion. One-hundredth  of  a  cubic  centimeter  of  virus  at  its  maximum  virulence  in- 
iected  into  a  locust  will  cause  the  characteristic  diarrhosa  in  two  hours  and  death 
an  hour  later.  This  method  of  increasing  the  virulence  takes  five  to  six  days  and 
this  period  of  time  has  to  be  taken  into  consideration  when  it  is  necessary  to  employ 
the  culture  on  a  practical  scale. 

When  the  acridian  to  be  infected  belongs  to  a  different  species  than  that  for  which 
the  virulence  of  B.  acridiorum  has  been  previously  augmented,  a  large  number  of 
passages  may  be  necessary  as  a  culture  virulent  for  one  species  may  not  be  able  to 
infect  another  species.  In  one  case  fifty-two  passages  were  necessary  in  order  to 
kill  Stauronautus  maroccanas  (Algeria)  in  eight  hours  while  for  the  same  insect  at 
Cyprus  only  twelve  passages  were  necessary.  It  is  also  desirable  that  the  first 
few  series  consist  of  a  large  number  of  insects,  as  there  will  be  apt  to  be  some 
which  will  be  more  sensitive  to  the  virus.  Their  natural  resistance  can  be  weakened 
by  fasting  for  several  days  before  inoculation.  The  intestinal  contents  should  not 
be  diluted  until  the  virus  will  kill  within  fifteen  hours. 

When  the  virulence  is  sufficiently  increased,  the  specific  bacillus  is  isolated  by 
means  of  an  agar  slant  or  plate  and  cultivated  for  twenty-four  to  thirty-six  hours 
at  room  temperature;  it  may  then  be  isolated  if  the  virus  is  to  be  conserved ;  if 
desired  for  direct  infection  experiments,  it  may  be  placed  dfrectly  in  broth.  The 
broths  used  is  made  as  follows: 


Water 1,000  c.c. 

Peptone 40  gr. 

Salt S  gr- 

Gelatin 30  gr. 

Dextrose S  gr. 


Boil,  alkalinize  slightly  and  filter;  place  in  bottles,  plug  with  cotton, 
cover  mouth  and  neck  with  parchment  paper  cap,  and  sterilize  at 
120°  for  thirty  minutes. 


The  gelatin  serves  to  fix  the  organisms  in  place  when  the  culture  is  sprayed  on  the 
vegetation,  and  on  account  of  the  dextrose  the  plants  are  greedily  devoured  by  the 
locusts. 


916  MICROBIAL  DISEASES    OF   INSECTS 

It  is  necessary  to  remember  that  the  virulence  of  B.  acridiorum  lowers  very  rapidly 
in  culture  and  is  attenuated  likewise  by  re-transplantations,  so  that  a  broth  culture 
so  prepared  should  be  used  within  two  or  three  days  at  the  utmost.  If  the  campaign 
against  the  locusts  lasts  for  several  months  or  the  regions  invaded  are  extensive, 
it  will  be  necessary  to  continue  the  series  of  passages  during  the  campaign  in  order 
to  have  on  hand  a  virus  of  maximum  efficiency.  It  is  best  to  make  two  or  three  more 
passages  than  necessary,  rather  than  too  few,  for  in  the  latter  case  the  results  of  a 
whole  campaign  may  be  nullified. 

The  material  necessary  for  a  campaign  against  the  locusts  consists  of  a  new 
spray  pump,  preferably  tinned  inside,  such  as  is  used  in  spraying  fruit  trees,  and  the 
bottles  containing  the  pure  broth  culture  of  B.  acridiorum  at  a  maximum  virulence. 
A  used  spray  pump  should  never  be  employed  as  it  is  practically  impossible  to  free 
it  from  the  antiseptic  contained.  The  pure  culture  should  be  used  as  soon  as  it 
shows  turbidity.  Broth  cultures  should  never  be  used  which  have  a  putrefactive 
odor. 

In  practice  it  is  generally  necessary  to  infect  the  greatest  number  of  locusts  in 
the  largest  possible  number  of  bands  in  order  to  exterminate  them  with  certainty 
in  as  short  a  time  as  possible.  The  quantity  of  broth  culture  to  be  sprayed  varies 
with  the  area  covered  by  the  nymphs  or  locusts.  One  liter  per  hectare  is  sufficient 
in  all  cases.  For  large  areas,  e.g.,  100-200  hectares,  spray  over  twenty  dif- 
ferent places,  using  one-half  liter  each  time,  taking  in  all  ten  liters.  It  is  better 
to  spray  over  a  large  area  rather  than  all  in  one  place,  choosing  places  where  the 
nymphs  or  locusts  are  in  largest  numbers,  and  always  spraying  the  type  of  plants 
preferred  and  in  advance  of  where  they  are  eating.  Spraying  should  be  done  in  the 
early  morning  or  preferably  in  the  evening  towards  sunset.  The  heat  and  especially 
the  bright  light  of  day  rapidly  attenuate  the  virulence  of  the  bacillus.  If  necessary 
to  spray  in  the  middle  of  the  day,  shady  spots  should  be  chosen. 

The  virulence  and  vitality  of  B.  acridiorum  has  been  conserved  in  the  dejecta 
and  dried  cadavers  for  seven  months  while  in  culture  the  virulence,  especially,  is 
lost  rapidly. 

A  very  hard  rain  will  inhibit  the  progress  of  an  epizootic  for  several  days.  The 
rain  washes  the  dejecta  of  the  locusts  from  the  contaminated  vegetation,  hindering 
this  mode  of  contagion;  the  epizootic  little  by  little  regains  its  normal  activity.  A 
rain  of  short  duration,  to  the  contrary,  seems  to  favor  the  progress  of  the  disease. 

A  cnrious  phenomenon  takes  place  when  a  band  of  infected  nymphs  meet  a  river 
in  the  course  of  their  route.  On  the  near  bank  is  found  an  actual  heap  of  cadavers, 
on  the  opposite  bank  likewise  but  they  are  very  much  less  in  number.  The  epi- 
zootic seems  to  be  completely  checked;  it  recommences  only  after  several  days  when 
it  takes  its  normal  course.  This  is  explained  by  the  fact  that  all  the  nymphs  al- 
ready badly  diseased  are  not  strong  enough  to  make  the  necessary  effort  to  cross  the 
stream  and  die  without  surmounting  the  obstacle;  those  which  were  only  slightly 
diseased  could  pass  it  but  were  so  enfeebled  by  their  effort  that  they  died  on  the 
opposite  bank.  Thus  the  colony  which  pursues  its  march  is  composed  only  of 
healthy  insects  and  of  several  nymphs  in  which  the  infection  has  hardly  begun. 

The  duration  of  an  epizootic  is  impossible  to  predict  for  all  species  of  insects  and 
nder  all  conditions;  as  a  general  rule  it  will  last  several  days,  most  often  several 


MICROBIAL  DISEASES    OF   INSECTS  917 

weeks,  rarely  several  months.  The  duration  of  an  epizootic,  however,  is  of  little 
importance;  the  object  is  to  cause  such  a  reduction  in  the  number  of  the  locusts  that 
these  insects  will  cease  to  be  a  plague. 

To  spread  the  epizootic  to  great  distances,  care  should  be  taken  to  infect  the 
winged  adults.  Some  species  of  locusts  are  more  sedentary  than  others,  it  follows 
that  the  more  sedentary  a  species  is,  the  more  necessary  to  multiply  the  foci  of 
infection. 

In  order  to  ascertain  whether  the  epizootic  is  progressing,  gather  one  hundred 
locusts  from  different  parts  of  the  swarm  and  by  pressing  their  abdomens,  see  how 
many  show  the  characteristic  diarrhoea.  Those  insects  showing  diarrhoea  one  day 
will  be  dead  the  next. 

Certain  peculiarities  were  observed  during  the  course  of  an  epizootic.  In  swarms 
infected  a  little  while  before  egg-laying,  numerous  females  lay  eggs  which  never 
reach  maturity;  others  never  reach  the  laying  stage  and  the  eggs  are  transformed 
within  the  body  to  a  blackish  mass.  Such  bands  were  annihilated  several  days 
afterward.  In  bands  of  nymphs  infected  several  days  before  the  last  molt  are  found 
numerous  abnormal  adults  with  poorly  developed  wings  only  half  their  ordinary 
length  which  prevent  them  from  flying,  and  further  a  microscopic  examination  of 
the  genital  organs  shows  complete  atrophy. 

SUSCEPTIBLE  INSECTS. — I.  Acridians. — B.  acridiorum  should  be 
pathogenic  for  all  acridians.  The  following  species  are  susceptible: 
Schistocerca  americana  (or  pollens). — Natural  epizootic  in  Yucatan  in 
1908-1911,  induced  in  the  Argentine  Republic  in  1912. 

Caloptenus  sp?. — Epizootic  induced  in  December  1912  in  the  region 
of  Rio  Negro,  Argentine  Republic. 

Stauronautus  maroccanus. — Epizootic  induced  in  1913  in  Algeria 
in  the  province  of  Oran,  and  in  the  isle  of  Cyprus. 

Schistocerca  paranensis  is  killed  by  B.  acridiorum.  (Argentine  Re- 
public.) 

Grylli*,s  pennsylvanicus ,  one  of  the  common  field  crickets  is  sus- 
ceptible. (DuPorte  and  Vanderleck). 

Zonocercus  elegans,  the  so-called  "elegant  grasshopper"  of  South 
Africa,  a  non-migratory  species,  was  used  in  inoculation  experiments 
with  this  bacillus.  It  was  concluded  that  this  disease  at  best  could 
be  employed  only  as  a  supplementary  measure  in  dealing  with  the  inva- 
sion of  these  insects  under  conditions  that  prevail  in  South  Africa. 

The  Philippine  locust,  Pachytylus  migrator -aides,  has  given  negative 
results  with  B.  acridiorum.  (Mackie). 

II.  ANTS.— A  species  of  small  ant  near  Paris  was  annihilated  in 
1911  by  B.  acridiorum. 


Ql8  MICROBIAL  DISEASES    OF   INSECTS 

Selenopsis  gemminata,  near  Buenos  Aires  was  annihilated  in  1912. 
Several  drops  of  the  culture  were  placed  in  each  ant  hill. 

A  tta  sexdens,  a  veritable  plague  in  the  tropical  and  sub-tropical 
countries  was  annihilated  at  Chaco  and  Tucuman  after  the  virulence 
had  been  increased  for  this  species  of  ant  by  many  passages. 

III.  CATERPILLARS. — A  field  of  cotton  which  was  being  ravaged  by 
caterpillars  was  treated  with  B.  acridiorum.     Four  days  afterward  all 
the  caterpillars  were  dead  while  a  neighboring  field,  treated  simul- 
taneously with  Paris  green,  still  contained  many  living  caterpillars. 

The  yellow  bear  caterpillar  (Spilosoma)  Diacrisia  mrginica)  has 
been  found  to  be  susceptible.  (DuPorte  and  Vanderleck). 

B.  acridiorum  does  not  attack  the  silk- worm,  Bombyx  mori;  it  kills 
the  cockchafer,  Melolontha  vulgaris,  by  injection  but  not  by  ingestion. 

Birds  and  mammals  in  general  are  immune  to  this  bacillus. 
One  notable  exception  is  the  sewer  rat  which  dies  from  generalized 
septicemia  a  few  hours  after  injection.  The  rat  was  immune  to  cul- 
tures ingested. 

IV.  BEETLES. — The  Colorado  potato  beetle,  both  larvae  and  adults, 
is  not  susceptible.     (DuPorte  and  Vanderleck). 

BACILLARY  SEPTICEMIA  OF  THE  CATERPILLARS  OF  Arctia  caja  L. 
Bacillus  cajce — Picard  and  Blanc* 

HISTORY  AND  DISTRIBUTION. — In  1913  the  vineyards  of  central 
France  were  almost  completely  destroyed  by  two  diseases;  one  of  these 
was  a  fungus  disease  caused  by  Empusa  aulica,  the  other  was  a  septi- 
cemia of  bacillary  origin. 

SYMPTOMS. — The  caterpillars  become  flaccid  and  emit  a  nauseating 
odor;  their  digestive  tube  contains  only  a  clear  liquid  free  from  all 
organisms.  The  blood  contains  a  pure  culture  of  a  bacillus  with  which 
the  disease  has  been  produced  artificially. 

CAUSAL  ORGANISM. — B.  caja  is  a  slightly  oval  bacillus,  about  1.5/1  in  length; 
motile;  Gram  negative;  stains  deeply  with  crystal  violet;  treated  by  Pappenheim's 
method  it  shows  a  characteristic  bi-polar  stain. 

*  Picard,  F.  and  Blanc,  G.  R.  On  a  bacillary  septicemia  of  caterpillars  of  Arctia  caja  L. 
Compt.  rend.  acad.  sci.  156,  1913,  pp.  1334-1336. 


MICROBIAL  DISEASES    OF   INSECTS  QIQ 

Broth  cultures  develop  in  twelve  hours  at  iS°-35°  with  an  optimum  of  25°;  from 
these  the  odor  of  H^S  is  perceptible;  in  twenty-four  hours  broth  cultures  have  a  green 
fluorescence  which  is  more  marked  at  2  5°  than  ati5°orat35°.  Grows  rapidly  on  both 
gelatin  and  agar  showing  a  green  fluorescence,  the  former  is  liquefied.  Growth  on 
potato  is  meager,  showing  only  after  forty-eight  hours  and  producing  no  pigment. 

METHODS  OF  INFECTION. — Artificial. — Caterpillars  of  Arctia  caja 
inoculated  in  one  of  their  feet  by  means  of  a  fine  needle  dipped  in  viru- 
lent blood  or  in  a  broth  culture,  die  regularly  in  three  days  at  15°, 
manifesting  in  their  blood  swarms  of  the  specific  bacteria.  If  kept  at 
25°  they  die  in  twelve  to  twenty-four  hours.  The  blood  of  the  cater- 
pillars kept  at  the  latter  temperature  appears  to  be  the  more  virulent. 

Caterpillars  receiving  several  drops  of  culture  by  means  of  a  pip- 
ette introduced  into  the  pharynx,  die  in  twelve  hours  at  25°  with  their 
blood  invaded  by  the  bacteria.  This  suggests  a  possible  practical 
application. 

SUSCEPTIBLE  INSECTS  AND  OTHER  ANIMALS. — Caterpillars  of  Por- 
thesia  chrysorrhea  are  very  sensitive  to  B.  caj(R  and  die  on  inoculation 
in  twenty-four  to  forty-eight  hours. 

The  following  Coleoptera:  Hydrophilus  pistaceus,  Dyticus  pisanus, 
Cybister  laterimarginalis,  Colymbetes  fuscm  are  not  killed  by  inocula- 
tion; nor  are  the  following  Hemiptera:  Notonecta  glauca,  Nipa  cinerea, 
Ranatra  linearis. 

The  white  rat  is  not  sensitive  to  intraperitoneal  injection  of  i  c.c.  of 
a  twenty-four-hour  broth  culture.  The  tree  frog,  Hila  arborea,  dies 
by  inoculation,  with  the  same  culture,  into  the  lymphatic  sacs  in 
twenty-four  to  forty-eight  hours  with  the  blood  invaded  by  numerous 
organisms.  The  blood  of  dying  caterpillars  is  more  virulent  for  the 
tree  frog  than  broth  cultures;  0.5  c.c.  injected  into  the  lymphatic 
sacs  causes  the  death  of  the  batrachian  in  twelve  hours  with  an 
intense  bacillary  septicemia. 

B.  caja  seems  to  belong  to  the  same  group  as  d'Herelle's  B.  acridio- 
rum.  It  is  distinguished  from  it  however  by  several  characteristics, 
both  biological  and  pathological,  being  a  parasite  of  the  blood  of  the 
caterpillars  whereas,  according  to  d'Herelle,  the  site  of  affection  in  the 
diseased  locusts  is  in  the  digestive  tract. 


Q20  MICROBIAL  DISEASES  OF  INSECTS 

GRAPHITOSIS* 
Bacillus  tracheitis  or  graphitosis — Krassilstschik 

HISTORY  AND  DISTRIBUTION. — This  disease  together  with  a  bac- 
terial septicemia  was  noted  among  the  Lamellicornia  in  1893  in  the 
southeast  of  Russia  by  Krassilstschik.  He  states  that  the  larvae  from 
the  Lamellicornia  which  formerly  died  en  masse  of  muscardine,f  die 
of  this  disease  very  seldom  in  recent  years.  Bacterial  parasites  seem 
to  have  replaced  it  in  this  part  of  Russia,  and  this  is  distinctly  advan- 
tageous since  the  bacterial  diseases  are  much  more  destructive  than 
any  of  the  species  of  muscardine. 

SYMPTOMS. — At  first  the  larva  is  entirely  pure  white,  then  several 
legs  change  to  a  bright  yellow  color,  next  to  a  yellow  brown.  Little 
by  little  this  coloration  extends  over  all  the  legs.  Later  on  both  sides 
of  the  larva,  characteristically  in  the  region  of  the  spiracles  and  around 
them,  the  skin  takes  on  a  grayish  hue  which  gradually  deepens.  The 
larva  is  generally  living  at  this  stage  but  appears  to  be  diseased.  This 
grayish  coloration  extends  toward  the  back,  and  the  anterior  part  of 
the  larva  then  gradually  becomes  gray  also.  At  this  stage  the  larva 
is  generally  dead.  After  death,  -the  gray  color,  spreading  characteris- 
tically from  the  spiracles,  deepens  considerably,  extending  all  over  the 
skin,  finally  acquiring  a  tint  resembling  that  of  polished  graphite, 
whence  its  name  "graphitosis."  This  coloration  is  very  characteristic 
for  the  larva  which  die  of  this  disease;  it  is  only  very  rarely  that  the 
cadaver  is  of  a  brownish  shade. 

When  the  infection  first  shows  in  the  legs,  the  movements  are  not 
inhibited  in  any  way,  but  when  the  graying  around  the  spiracles  sets 
in,  the  larva  becomes  comatose  yet  still  responding  to  exterior  excita- 
tions. It  soon  dies,  retaining  its  characteristic  curve  but  gradually 
becoming  limp  and  soft  and  decreasing  in  size,  length,  etc. 

CAUSAL  ORGANISM. — The  bacillus  of  graphitosis  is  from  2/t  to  2.2/z  long  having  a 
diameter  of  more  than  half  its  length;  spores  are  produced;  very  motile,  movements 
quick  and  rapid;  occurs  generally  in  pairs  from  3.6/1  to  4.6/4  long;  long  filaments  not 
produced;  the  longest  do  not  exceed  7/1  to  9/1  which  corresponds  to  two  pairs  of  bacilli 

*Bacillary  Diseases  of  Lamellicornia. — In  1893  Krassilstschik  described  two  bacterial 
diseases  attacking  the  larvae  of  the  following  insects:  Rhizolrogus  solstilialis,  Melolonlha 
vulgaris,  Anisoplia  austriaca,  crucifera,  and  fruticola,  Cetonia  sp.  and  a  larva  belonging  to 
the  Geotrupini  (Lethrusf  sp.). 

t  Krassilstschik,  I.  Graphitosis  and  Septicemia  of  Insects.  Memoires  Soc.  Zool.  en 
France,  t.  vi,  pp.  245-285,  1893. 


MICROBIAL   DISEASES    OF   INSECTS  Q2I 

end  to  end.     Aerobic;  shrinks  perceptibly  when  treated  with  Gram's  stain,  almost 
to  half  its  size.     Stained  bacilli  show  unstpined  spots  (spores). 

In  plate  cultures  B.  tracheitis  develops  into  small  circular  colonies  0.25  to  0.75 
mm.  in  diameter,  which  are  covered  with  tubercles  when  the  colony  grows  in  gela- 
tiu.  Gelatin  colonies  are  finely  granular  of  a  deep  brownish-yellow  color,  opaque 
center  surrounded  by  a  transparent  ring.  Gelatin  is  liquefied  in  twenty-four  to 
forty-eight  hours.  Gelatin  stab  cultures  are  typical,  having  a  cup-shaped  hollow 
funnel  at  the  surface,  a  short  empty  stem;  then  the  culture  grows  in  the  depth  of 
the  gelatin,  liquefying  it  in  the  shape  of  a  carrot,  later  becoming  the  shape  of  an 
inverted  bottle;  the  culture  is  seen  along  the  original  path  of  the  stab  as  a  zigzag 
line  which  later  forms  a  compact  cream-colored  deposit  as  the  gelatin  becomes 
entirely  liquefied;  these  cultures  have  the  odor  of  the  white  of  an  egg.  Broth 
cultures  are  clear  the  first  twenty-four  hours;  after  that  they  become  turbid  and 
a  pellicle  forms  which  thickens  with  age,  sediment  compact;  cultures  become 

wholly  transparent  in  four  to  six  months. 

• 

METHODS  OF  INFECTION. — Washing  the  larvae  of  the  Lamellicornia 
with  the  natural  virus  of  graphitosis  kills  16.6  per  cent  to  100  per  cent 
but  an  augmented  virus  gives  100  per  cent  mortality.  The  injection 
under  the  skin  of  a  very  small  drop  of  graphitosis  blood  is  always  fatal 
for  the  larva  even  if  the  virus  is  weak. 

B.  tracheitis  multiplies  first  in  the  blood  system,  then  fills  the 
Malpighian  tubes,  next  characteristically  in  the  trachea  and  then  in  the 
fatty  bodies;  the  trachea  becomes  typically  filled  with  black  amorphous 
granules  from  whence  the  name  of  this  organism;  the  fat  cells  are  at- 
tacked. The  intima  of  the  muscles,  trachea  and  other  organs  is  covered 
with  bacilli,  which  however  do  not  penetrate  the  organs  themselves. 

AMERICAN  FOUL  BROOD 
Bacillus  larva — White* 

HISTORY  AND  DISTRIBUTION. — American  foul  brood  is  the  prevalent 
disease  among  bees  in  America  and  is  distributed  through  all  parts 
of  the  United  States,  in  Ontario,  Canada,  Switzerland,  New  Zealand, 
Germany,  England,  and  France  and  it  is  probable  that  it  has  a  much 
wider  geographical  distribution. 

SYMPTOMS. — American  foul  brood  or  simply  "foul  brood"  usually 
shows  itself  in  the  larva  just  about  the  time  that  the  larva  fills  the  cell 
and  after  it  has  ceased  feeding  and  has  begun  pupation. 

At  this  time  it  is  sealed  over  in  the  comb.     The  first  indication  of 

*  White,  G.  P.     American  foul  brood.  Bui.  809.  Bur.  of  Ent.  U.  S.  Dept.  of  Agr.,  1920. 


Q22  MICROBIAL   DISEASES    OF   INSECTS 

the  infection  is  a  slight  brownish  discoloration  and  the  loss  of  the  well- 
rounded  appearance  of  the  normal  larva.  At  this  stage  the  disease  is 
not  usually  recognized  by  the  beekeeper.  The  larva  gradually  sinks 
down  in  the  cell  and  becomes  darker  in  color,  and  the  posterior  end  lies 
against  the  bottom  of  the  cell.  Frequently  the  segmentation  of  the 
larva  is  clearly  marked.  By  the  time  it  has  partially  dried  down  and 
become  quite  dark  brown  (coffee  colored)  the  most  typical  character- 
istic of  this  disease  manifests  itself.  If  a  match  stick  or  tooth-pick  is 
inserted  into  the  decaying  mass  and  withdrawn  the  larval  remains  ad- 
here to  it  and  are  drawn  out  into  a  thread,  which  sometimes  extends  for 
several  inches  before  breaking. 

This  ropiness  is  the  chief  characteristic  used  by  the  beekeeper  in 
diagnosing  this  disease.  The  larva  continues  to  dry  down  and  gradually 
loses  its  ropiness  until  it  finally  becomes  a  mere  scale  on  the  lower  side 
wall  and  base  of  the  cell. 

The  scale  formed  by  the  dried-down  larvae  adheres  tightly  to  the 
cell  and  can  be  removed  with  difficulty  from  the  cell  wall.  The  scales 
can  best  be  observed  when  the  comb  is  held  with  the  top  inclined  to- 
ward the  observer  so  that  a  bright  light  strikes  the  lower  side  wall.  A 
very  characteristic  and  usually  penetrating  odor  is  often  noticeable  in 
the  decaying  larvae.  This  can  perhaps  best  be  likened  to  the  odor  of 
heated  glue. 

The  majority  of  the  larvae  which  die  of  this  disease  are  attacked 
after  being  sealed  in  the  cells.  The  cappings  are  often  entirely  removed 
by  the  bees,  but  when  they  are  left  they  usually  become  sunken  and 
frequently  perforated.  As  the  healthy  brood  emerges  the  comb  shows 
the  scattered  sunken  cappings  covering  dead  larvae,  giving  it  a  char- 
acteristic appearance. 

Pupae  also  may  die  of  this  disease,  in  which  case  they  too,  dry  down, 
become  ropy,  and  have  the  characteristic  odor  and  color.  The 
tongue  frequently  adheres  to  the  upper  side  wall  and  often  remains  there 
even  after  the  pupa  has  dried  down  to  a  scale.  Younger  unsealed  larvae 
are  sometimes  affected.  Usually  the  disease  attacks  only  worker 
broods,  but  occasional  cases  are  found  in  which  queen  and  drone  broods 
are  diseased.  It  is  not  certain  that  race  of  bees,  season,  or  climate 
have  any  effect  on  the  virulence  of  this  disease,  except  that  in  warmer 
climates  where  the  breeding  season  is  prolonged,  the  rapidity  of  devas- 
tation is  more  marked. 


MICROBIAL  DISEASES   OF   INSECTS  923 

CAUSAL  ORGANISM.—  Bacillus  larva  is  a  slender  rod  with  ends  slightly  rounded 
and  with  a  tendency  to  grow  in  chains.  The  length  varies  greatly,  depending  for 
the  most  part  upon  the  medium  used  for  its  cultivation.  It  varies  from  2.5  to  SM 
in  length,  and  is  about  0.5/1  in  breadth  when  grown  on  the  surface  of  brood-filtrate 
agar.  In  a  liquid  medium  it  is  usually  much  longer,  frequently  becoming  filamen- 
tous. Giant  whips  occur  in  large  numbers,  especially  in  the  condensation  water  of 
brood-filtrate  agar  slant  cultures.  They  are  also  present  in  decaying  larvae  dead  of 
American  foul  brood.  The  flagella  are  peritrichic:  when  twisted  into  giant  whips, 
these  corkscrew-like  structures  vary  widely  in  their  dimensions  from  scarcely  visible 
coiled  filaments  to  bodies  several  microns  in  diameter.  Motility  moderate  in  young 
cultures  from  the  surface  of  brood-filtrate  agar,  sluggish  in  liquid  cultures.  Spores 
formed  about  the  third  day  on  brood-filtrate  agar;  median,  causing  a  spindle-shaped 
enlargement  of  the  rod;  free  spores  measure  about  0.6  by  1.3^.  Few  or  no  spores 
are  formed  in  liquid  media,  deep  in  solid  media,  and  on  media  containing  glycerin, 
mannit,  or  dextrose.  Some  of  the  other  sugars,  and  also  honey  inhibit  spore  forma- 
tion. The  rods  stain  readily  with  ordinary  anilin  dyes,  and  are  Gram  positive. 

B.  larva  is  cultivated  with  difficulty,  growing  best  on  media  made  as  the  ordinary 
laboratory  media,  substituting  bee  larvae  for  meat,  or  on  egg-yolk-suspension  agar. 
Bee  larvae  agar,  however,  is  limited  in  its  usefulness  on  account  of  the  large  amount  of 
brood  required  in  its  preparation.  The  unheated  egg-yolk  agar  is  prepared  as 
follows:  immerse  fresh  eggs  in  a  disinfecting  solution,  break  the  shell,  pour  off  the 
white,  and  drop  the  yolk  into  a  flask  containing  about  70  cc.  of  sterile  water;  agi- 
tate the  flask  to  make  a  homogeneous  suspension  of  the  yolk,  and  with  a  sterile 
pipette  transfer  the  aqueous  suspension  to  sterile  tubes  and  store  until  needed.  For 
use :  melt  tubes  of  agar  and  cool  to  about  50°,  add  about  i  cc.  of  egg-yolk-suspension 
to  each  5  c.c.  of  melted  agar,  and  either  incline  and  allow  to  harden,  or  use  im- 
mediately for  plating  as  desired.  A  more  detailed  description  of  the  technic  em- 
ployed in  making  both  of  the  special  media  necessary  for  the  cultivation  of  B.  larva 
will  be  found  in  the  bulletin  by  White.  Bacillus  larva  is  present  in  practically  pure 
cultures  in  brood  dead  of  American  foul  brood,  so  this  organism  can  be  readily  ob- 
tained from  brood  dead  of  this  disease  by  heating  the  spore-containing  material  in 
aqueous  suspension  at  100°  for  one  or  two  minutes,  and  plating  in  bee-larvae  agar  or 
egg-yolk  suspension  agar.  When  bee-larvae  agar  alone  is  employed  and  inoculations 
are  made  with  spores,  following  Liborious'  method  for  anaerobes,  growth  as  a  rule 
appears  more  often  near  to  than  on  the  surface,  indicating  partial  anaerobiosis. 
Sub-cultures  on  brood-filtrate  agar  and  egg-yolk-suspension  agar  or  their  combina- 
tion yield  abundant  surface  growth. 

Brood-filtrate  agar  slant,  growth  rapid,  being  moderate  to  heavy  in  twenty-four 
hours,  somewhat  spreading,  grayish  white  and  slightly  viscid;  has  a  more  or  less 
uniform  border,  a  smooth  surface,  and  a  ground  glass  appearance.  Older  cultures 
are  less  prominent  than  the  younger  ones.  Brood-filtrate  agar  plates,  surface  colonies 
vary  in  size  depending  upon  the  number  present.  When  well  isolated  they  not  in- 
frequently spread,  attaining  a  diameter  of  i  cm.  or  more;  growth  only  slightly  raised, 
smooth  surface,  ground  glass  appearance,  with  a  clearly  defined,  uniform  border. 
Deep  colonies  vary  from  lenticular  to  irregular  in  form  with  filamentous  outgrowths 
from  portions  of  their  surface.  No  visible  gas,  but  slight  acidity  in  carbohydrate 


924  MICROBIAL  DISEASES    OF   INSECTS 

broths  to  which  a  little  brood  filtrate  or  egg  suspension  has  been  added.  Gelatin, 
no  growth  in  plain  or  in  brood-filtrate  gelatin  at  temperatures  at  which  it  remains 
solid. 

The  more  resistant  spores  of  B.  larva  require  100°  for  eleven  minutes  to  destroy 
them,  and  when  suspended  in  hone>  require  a  half  hour  or  more.  Five  per  cent, 
carbolic  acid  is  resisted  for  months,  i-iooo  mercuric  chloride  for  days,  10  per  cent, 
formalin  for  hours,  and  20  per  cent,  formalin  for  thirty  minutes,  in  each  case  at 
room  temperature.  In  fact,  most  destructive  agencies  are  resisted  by  these  spores. 
Drying  at  room  temperature  has  been  resisted  for  nine  years,  and  it  is  most  likely 
that  they  will  remain  viable  and  virulent  for  a  very  much  longer  period.  Dry 
spores  exposed  to  direct  sunlight  are  killed  in  from  twenty-eight  to  forty-one 
hours.  Four  to  six  weeks  are  required  for  destruction  when  suspended  in  honey 
and  exposed  to  the  direct  rays  of  the  sun,  but  if  shielded  from  direct  sunlight,  the 
spores  remain  alive  and  virulent  for  more  than  a  year.  The  destructive  effects  of 
fermentation  have  been  resisted  for  more  than  seven  weeks  at  incubator  and  out- 
door temperatures,  and  it  is  likely  that  a  much  longer  period  could  have  been 
withstood. 

B.  larva  is  pathogenic  for  the  larval,  prepupal,  or  early  pupal  stages  of  the  brood 
of  honey  bees.  Adult  bees,  rabbits,  guinea  pigs,  rats  and  humans  are  not  suscepti- 
ible  to  infection. 

METHODS  OF  INFECTION. — Natural. — American  foul  brood  infec- 
tion is  transmitted  primarily  through  the  food  of  bees;  possibly  at 
times  to  some  extent  through  their  water  supply.  Robbing  from  the 
diseased  colonies  of  the  apiary,  or  from  neighboring  apiaries,  is  the 
most  likely  mode  by  which  the  disease  is  transmitted  in  nature.  The 
placing  of  brood  combs  containing  diseased  brood  with  healthy  colonies 
will  also  result  in  the  transmission  of  the  disease.  It  is  not  likely  that 
infection  ever  occurs  through  the.  medium  of  flowers.  Queens  and 
drones  have  been  presumably  overestimated  at  times  as  possible  sources 
of  infection.  It  has  not  been  determined  as  yet  whether  American 
foul  brood  is  ever  transmitted  by  them.  The  clothing  or  hands  of 
those  about  an  apiary  or  handling  the  bees  are  not  fruitful  sources  for 
the  transmission  of  the  disease.  The  hive  tool,  if  brought  in  direct 
contact  with  dead  larvae  in  testing  for  the  presence  of  disease,  might 
serve  to  transmit  infection,  but  during  the  usual  manipulation  it  would 
not.  Other  tools  and  bee  supplies  generally  about  an  infected  apiary 
will  not  transmit  the  infection  in  the  absence  of  robbing  from  those 
sources. 

Artificial.— American  foul  brood  can  be  communicated  by  feeding 
to  a  healthy  colony  the  scales  from  combs  which  had  contained  brood 


MICROBIAL  DISEASES    OF   INSECTS  925 

affected  with  American  foul  brood;  likewise  when  these  scales  are  placed 
in  ordinary  meat  broth,  incubated  twenty-four  hours  and  then  heated 
to  65°  for  twenty  minutes;  infection  in  this  case  is  due  to  the  presence 
of  spores.  Pure  cultures  of  B.  larva  mixed  with  sterile  sugar  sirup  and 
fed  to  healthy  colonies  produce  the  disease  within  three  weeks.  B. 
larva  can  be  obtained  in  pure  culture  from  such  diseased  larvae. 

A  fact  of  special  importance  not  only  in  the  technic  of  making 
studies  but  also  in  the  control  of  the  disease  is  that  colonies  in  which 
the  disease  has  been  produced  through  artificial  inoculation  can  be 
kept  in  the  experimental  apiary  without  transmitting  the  disease  to 
others. 

B.  larva  may  be  obtained  in  large  quantities  suitable  for  experi- 
mental inoculation  by  diluting  and  filtering  the  crushed  bodies  of 
bee  larvae  through  a  Berkefeld  or  other  fine  filter. 

CONTROL. — The  treatment  of  an  infectious  bee  disease  consists 
primarily  in  the  elimination  or  the  removal  of  the  cause  of  the  disease. 
Effort  is  not  made  to  save  the  larvae  already  dead  or  dying,  but  to  stop 
further  devastation  by  removing  all  material  capable  of  transmitting 
the  cause  of  the  trouble.  The  swarm  is  transferred  from  the  infected 
hive  to  a  clean  disinfected  hive;  the  infected  combs  from  the  old  hive 
should  either  be  burned,  melted,  or  boiled  thoroughly  before  the  wax  is 
fit  for  use  again.  The  honey  taken  from  the  infected  hive  should  be 
buried  or  at  least  removed  so  that  no  bees  can  use  it  for  food.  This 
treatment  may  have  to  be  repeated  before  the  disease  is  under  control. 
Brood  from  badly  diseased  colonies  should  be  burned,  buried  or  other- 
wise destroyed  at  once.  Combs  even  if  they  appear  white  and  clean 
should  be  melted.  Chemical  disinfectants  should  not  be  relied  upon. 
Infected  hives  should  be  burned  over  inside  with  a  gasoline  or  oil  torch. 

SEPTICEMIA  OF  THE  COCKCHAFER,  Melolontha  vulgaris 
Bacillus  melolontha — Chatton* 

HISTORY. — In  May,  1912,  while  studying  the  effect  of  d'Herelle's 
B.  acridiorum  on  the  cockchafer,  Chatton  noticed  that  the  cockchafers 
were  dying  from  a  spontaneous  septicemia;  this  he  found  later  was  due 
to  a  coccobacillus  which  he  named  B.  melolontha. 

SYMPTOMS. — No  symptoms  are  noted. 

*  Chatton,  E.:  Spontaneous  septicemia  in  the  cockchafer  and  the  silk  worm  due  to  cocco- 
bacilli.     Compt.  rend.  acad.  sci.  156,  1913.  PP.  1707-1709. 


926  MICROBIAL  DISEASES   OF  INSECTS 

CAUSAL  ORGANISM. — B.  melolontha  resembles  B.  acridiorum  of  d'Herelle  with  the 
exception  of  the  following  characteristics:  the  bacillus  is  longer,  and  in  agar  culture 
produces  a  green  fluorescence  in  five  to  six  days.  It  is  distinguished  from  the  bacillus 
of  d'Herelle  in  addition  by  its  pathogenic  action  on  the  silk- worm,  Bombyx  mori. 

METHODS  OF  INFECTION. — Injected  into  the  general  cavity,  B. 
melolonthce  kills  the  cockchafer  in  twelve  to  thirty-six  hours,  and  where 
its  virulence  has  been  augmented  by  several  passages  through  this 
insect,  always  in  less  than  twenty-four  hours,  but  per  os,  it  is  as 
inactive  as  B.  acridiorum.  Seventy-five  per  cent  of  healthy  cock- 
chafers show  the  presence  of  B.  melolonthce  in  their  digestive  tube, 
sometimes  in  massive  culture.  This  is  always  the  case  with  cock- 
chafers affected  with  septicemia. 

This  blood  disease  seems  to  be  of  intestinal  origin  however,  as  with 
the  locust.  B.  melolonthce,'  a  common  parasite  of  the  intestine  of  the 
cockchafer  passes  into  the  general  cavity  only  under  special  conditions 
yet  unknown.  When  this  organism  is  removed  from  the  intestine  and 
injected  into  the  general  cavity,  septicemia  is  produced. 

It  is  as  virulent  for  the  silk-worm  by  injection  as  for  the  cockchafer, 
and  as  inactive  by  ingestion. 

EUROPEAN  FOUL  BROOD 
Bacillus  pluton— White* 

HISTORY  AND  DISTRIBUTION. — This  type  of  foul  brood,  sometimes 
known  as  "black  brood,"  or  "New  York  bee  disease"  is  not  nearly  as 
wide  spread  in  the  United  States  as  is  American  foul  brood,  but  in  cer- 
tain parts  of  the  country  it  has  caused  enormous  losses.  It  is  spread 
over  England,  Germany,  Switzerland  and  other  parts  of  Europe  and 
has  been  noted  many  times  during  the  last  decade. 

SYMPTOMS. — The  presence  of  disease  can  usually  be  detected  in  an 
experimental  colony  during  the  week  that  feeding  is  begun.  The  first 
indication  of  it  may  be  that  only  a  portion  of  a  larva  is  seen  in  a  cell, 
the  remaining  portion  having  been  removed  by  the  bees.  Aside  from 
an  observation  of  this  kind,  the  earliest  indication  one  gets  from  the 
macroscopic  examination  is  that  sick  larvae  are  found  among  the  un- 
capped brood. 

Sick  larvae  manifest  certain  symptoms  during  the  course  of  the 
disease  by  which  its  presence  can  be  diagnosed  while  the  larvae  are  still 

*  White  G.  P.:  European  foul  brood.    Bui.  810.  B.  of  Ent.  U.  S.  Dept.  of  Agr.  1920. 


MICROBIAL  DISEASES   OF   INSECTS  927 

alive.  The  length  of  time  that  a  developing  bee  is  sick  of  European 
foul  brood  is  variable.  In  general,  the  three  days  just  preceding  the 
time  when  a  larva  would  ordinarily  be  capped,  is  the  most  favorable 
period  for  making  a  diagnosis  from  the  gross  examination  alone. 
Healthy  larvae  at  a  certain  age  when  slightly  magnified  show  a 
peristalsis-like  motion  of  their  bodies,  but  larvae  of  this  same  age 
when  sick  frequently  exhibit  a  marked  peristalsis  which  can  easily  be 
seen  with  the  unaided  eye.  Diseased  larvae  may  show  a  yellowish 
tint  or  appear  transparent  instead  of  the  glistening  white  or  bluish 
white  of  healthy  larvae. 

Another  symptom  often  serves  for  diagnosis.  In  a  healthy  larva  a 
pollen-colored  mass  is  frequently  plainly  seen  through  the  transparent 
area  along  the  dorsal  median  line.  If  this  intestinal  mass  appears 
white  or  yellowish  white,  the  presence  of  European  foul  brood  is  al- 
most certain.  This  may  be  often  more  plainly  observed  if  the  larva 
is  removed  from  the  cell  with  forceps. 

European  foul  brood  may  be  positively  diagnosed  in  living  larvae 
of  a  favorable  age  and  condition  by  the  following  method :  Remove  the 
larva  to  be  tested  from  the  cell  and  place  it  upon  glass,  preferably  with 
a  dark  background;  with  a  dissecting  needle  in  each  hand  and  with 
their  points  near  together,  pierce  with  both  needles  so  as  to  tear  the 
body  wall  crosswise,  and  continue  to  separate  the  two  portions  of  the 
larva.  If  the  larva  is  diseased,  and  one  is  successful,  it  will  be  found 
that  the  intestinal  content  will  be  stripped  from  and  pulled  out  of  the 
posterior  and  blind  end  of  the  canal.  The  intestinal  content  of  healthy 
living  larvae  cannot  be  removed  in  this  way.  The  force  which  is  ap- 
plied in  pulling  the  mass  from  the  intestine  frequently  causes  the  typ- 
ical transparent,  mucus-like  substance  surrounding  the  central  mass  to 
stretch  and  the  enclosed  whitish  substance  to  break  into  segments; 
this  appearance  is  very  characteristic. 

If  the  disease  is  more  advanced,  a  portion  of  the  intestinal  content 
may  flow  out  in  the  form  of  a  sac,  the  wall  of  which  is  very  easily  bro- 
ken. When  broken  the  content  of  this  sac-like  structure  will  flow  out 
as  a  rather  thin  whitish  or  yellowish  white  fluid  containing  small  whitish 
granules  that  vary  in  size.  If  the  disease  is  far  advanced  and  the  larva 
probably  dead,  the  enveloping  substance  of  the  intestinal  content  is 
so  easily  broken  that  often  only  the  whitish  or  yellowish-white  fluid 
flows  from  the  ruptured  wall  of  the  larva. 


928  MICROBIAL  DISEASES   OF   INSECTS 

Dying  larvae  diseased  with  European  foul  brood  frequently  show 
the  segments  of  the  body  marked  off  less  distinctly  than  living  healthy 
larvae. 

CAUSAL  ORGANISM. — B.  pluton,  the  organism  of  European  foul  brood,  is  a  small, 
non-spore-forming  organism,  sharply  pointed  at  one  or  both  ends,  about  i/i  long  and 
less  than  o.5ju  in  breadth,  on  the  average;  occurs  frequently  in  pairs;  single  individuals 
vary  very  markedly  in  size  and  shape. 

This  organism  has  never  been  cultivated,  but  sections  of  larvae  in  various  stages 
of  the  disease  show  B.  pluton  to  be  the  first  invader  of  healthy  larvae.  B.  pluton 
gains  entrance  to  the  larva  by  way  of  the  mouth.  The  growth  and  multiplication  of 
the  parasite  take  place  within  the  stomach  and  do  not,  during  the  life  of  the  larva 
get  beyond  the  peritrophic  membrane.  The  tissues  therefore,  are  not  invaded  by  it. 
The  secondary  invaders  in  European  foul  brood,  B.  ahei,  Strept.  apis,  Bad.  eury- 
dice,  and  B.  orpheus,  rarely,  if  ever,  invade  the  tissues  until  the  larva  is  dead  or  nearly 
so.  In  American  foul  brood,  practically  speaking,  there  are  no  secondary  invaders, 
either  during  the  life  of  the  infected  larva,  or  during  the  decay  of  the  remains. 

Experimentally,  B.  pluton  suspended  in  water,  was  killed  at  approximately  63° 
in  ten  minutes,  but  when  suspended  in  honey,  79°  for  ten  minutes  had  to  be  ap- 
plied. Dried,  B.  pluton  remained  alive  and  virulent  for  approximately  a  year.  In 
the  dry  state  direct  sunlight  was  not  destructive  until  after  twenty-one  to  thirty-one 
hours,  but  when  suspended  in  water,  only  five  to  six  hours  were  required  for  de- 
struction, and  when  suspended  in  honey  exposure  for  from  three  to  four  hours  was 
fatal. 

In  the  presence  of  fermentative  processes  in  a  10  per  cent  sugar  solution  B. 
pluton  was  destroyed  in  from  three  to  five  days  at  incubator  temperature  and  in 
from  eleven  to  twenty-one  days  at  room  temperature.  In  a  fermenting  honey 
solution  outdoors,  it  was  still  alive  and  virulent  after  one  month;  in  undiluted 
honey  at  room  temperature  B.  pluton  ceased  to  be  virulent  in  from  three  to  seven 
months.  Mixed  with  pollen,  this  bacillus  remained  alive  and  virulent  for  more  than 
seven  months  at  room  temperature  and  for  more  than  ten  months  at  refrigerator 
temperature.  Putrefactive  processes  were  destructive  to  B.  pluton  in  from  seven  to 
thirteen  days  at  incubator  temperature,  in  from  twenty-one  to  thirty-five  days  at 
room  temperature,  while  at  outdoor  temperature  it  remained  alive  and  virulent 
for  more  than  forty  days,  the  maximum  time  not  being  determined. 

B.  pluton  was  destroyed  by  0.5  per  cent  carbolic  acid  solution  in  from  eight  to 
eighteen  days;  i.o  per  cent  required  only  five  hours  to  four  days,  while  2  and  4  per 
cent  solutions  required  less  than  six  hours. 

Neither  man,  the  common  experimental  animals,  nor  insects  other  than  honey 
bees,  so  far  as  is  known,  are  susceptible  to  infection  with  the  European  foul  brood 
bacillus. 

METHODS  OF  INFECTION  %AND  CONTROL. — These  are  essentially  the 
same  as  those  for  American  foulbrood. 


MICROBIAL  DISEASES  OF  INSECTS  Q2Q 

BACTERIAL  SEPTICEMIA  OF  LARV.E  OF  THE  LAMELLICORN.E 
Bacillus  septicus  insectorum — Krassilstschik 

HISTORY  AND  DISTRIBUTION. — This  disease  occurred  separately 
and  together  with  graphitosis  previously  described. 

SYMPTOMS. — The  septicemia  produced  in  larvae  of  the  Lamelli- 
cornae  by  B.  septicus  insectorum  is  characterized  by  a  uniform  browning 
of  the  body  of  the  larvae.  As  death  approaches,  the  larva  shrivels  up 
and  when  dead  is  about  half  its  natural  size  and  is  of  a  deep  brown  color. 
During  the  progress  of  the  disease,  the  larva  ejects  a  very  black,  abund- 
ant, viscous,  semifluid  substance  from  the  anus  which  soils  the  ex- 
tremity of  the  abdomen. 

CAUSAL  ORGANISM. — This  bacillus  is  1.2/1  to  i.Sji  long  and  from  o.6ju  to  o.g/x  in  di- 
ameter; often  in  pairs;  long  filaments  not  formed.  Spores  are  characteristically  dip- 
lospores  although  isolated  ones  occur  not  infrequently.  It  shrinks  very  little  when 
stained  by  Gram's  method. 

Gelatin  is  liquefied;  subsurface  colonies  are  decidedly  lemon  shaped,  yellowish 
brown,  finely  granular,  surface  of  colony  typically  curled.  Surface  colonies  are  con- 
centrically three-ringed,  the  interior  opaque,  the  second  ring  more  transparent,  the 
third  very  thin  and  finely  granular.  Saccate  liquef action  in  gelatin  stab;  the  gelatin 
is  blackened  and  has  a  very  disagreeable  odor.  Spores  are  found  in  the  sediment  at 
the  bottom  of  the  tube.  Broth  is  made  turbid  in  eighteen  to  twenty  hours,  no 
pellicle,  bad  odor. 

METHODS  OF  INFECTION. — Healthy  larvae  inoculated  with  B.  sep- 
ticus insectorum  by  placing  cotton  saturated  with  a  broth  culture  on  a 
wound,  died  in  most  cases  with  typical  symptoms. 

The  sole  habitat  of  these  microbes  before  death  is  in  the  blood 
system. 

BACTERIAL  DISEASE  OF  THE  GUT-EPITHELIUM  OF  Arenicola 

ecaudata,  the  Lug- Worm 
Bacterium  arenicola. — Fantham  and  Porter.* 

HISTORY  AND  DISTRIBUTION. — This  bacillus  was  found  in  the  lumen 
of  the  gut  and  within  the  intestinal  epithelium  of  specimens  of  Areni- 
cola ecaudata  obtained  from  Plymouth,  England.  This  disease  is  not  of 
frequent  occurrence. 

*  Pantham,  P.  B.  and  Porter,  A.     Bacillus  arenicolct,  n.  sp.t  a  pathogenic  bacterium  from  the 
gut-epithelium  of  Arenicola  ecaudata.     Cent.  f.  Bakt.  I.,  Orig.  52,  1909,  320-334- 
59 


930  MICROBIAL  DISEASES    OF   INSECTS 

SYMPTOMS. — No  external  symptoms  are  noted.  Lesions  are  pro- 
duced in  the  epithelium,  the  cells  undergoing  degeneration,  perhaps 
shortening  the  life  of  the  lug- worm.  Bad.  arenicola  seems  to  be  con- 
fined chiefly  to  the  ciliated  tracts,  as  was  determined  by  microscopical 
examination  of  sections. 

METHODS  OF  INFECTION. — No  methods  of  infection  are  noted. 
From  the  type  of  the  disease,  however,  infection  per  os  is  suggested. 

CAUSAL  ORGANISM. — Bacterium  arenicolcs  averages  about  n/i  long  and  IM 
broad.  Extreme  individuals  measure  7/4  to  i7/x  long  by  0.7/1  to  1.3;*  broad;  some  of 
the  larger  forms  are  slightly  sinuous  in  outline;  no  flagella;  chromatophile  granules 
determined  by  staining  with  iron-hematoxylin,  are  present,  often  in  considerable 
numbers,  scattered  through  the  cell;  these  granules  are  often  concentrated  into 
transverse  bars  both  of  which  in  some  specimens  are  refractile.  The  cytoplasm 
stains  with  difficulty  with  plasma  stains.  Division  is  transverse.  One  terminal 
spore  is  formed  which  does  not  cause  the  enlargement  of  the  rod  to  any  extent. 
No  cultural  characteristics  are  given. 

IMPORTANCE. — This^disease  is  of  no  special  economic  importance. 

PSEUDOGRASSERIE    OF    THE    GlPSY-MOTH    CATERPILLAR 

Bacillus  lymantricola  adiposus — Paillot 

HISTORY  AND  DISTRIBUTION. — In  August,  1917,  Pa illot(  France) 
noted  a  gipsy  moth  caterpillar  which  presented  exterior  symptoms  of 
both  grasserie  and  flacherie.  It  was  infected  by  two  coccobacilli  to 
which  he  gave  the  names  Bacillus  lymantricola  adiposus  and  Bacillus 
lymantricz  j3.  The  former  bacillus  was  the  sole  cause  of  the  disease  in 
question  as  was  proved  by  experimentation.  The  following  season 
was  so  dry  that  epizootics  were  rare  among  insects  and  no  new  cases 
were  observed. 

SYMPTOMS. — A  few  hours  after  inoculation  with  B.  lymantricola 
adiposus  the  blood  of  the  caterpillar  possessed  the  same  milky  appear- 
ance as  that  of  caterpillars  affected  with  grasserie.  This  is  due,  not  to 
polyhedral  bodies  as  in  true  grasserie,  but  to  the  presence  of  fat  glo- 
bules in  the  blood.  The  name  pseudograsserie  is  given  to  this  disease 
as  it  resembles  grasserie  as  to  external  symptoms  only. 

CAUSAL  ORGANISM. — B.  lymantricola  adiposus  is  a  coccobacillus  i/j.  in  diameter  and 
2/j.  long;  strains  well,  showing  bi-polar  staining.  In  the  blood  of  the  caterpillars 
of  Lymantria  dispar,  this  organism  shows  most  unexpected  forms;  e.g.,  giant  forms, 
made  up  of  a  more  or  less  round  mass  whose  diameter  may  be  7  to  8/u,  and  greatly 


MICROBIAL  DISEASES   OF   INSECTS  931 

elongated  bacillus  forms,  facing  or  more  or  less  near  this  mass,  of  variable  lengths 
(cells  30  to  40/1  long  have  been  observed);  the  round  masses  have  also  been  ob- 
served without  the  elongated  bacillus  forms.  These  giant  forms,  true  forms  of 
growth,  are  only  met  with  during  the  earlier  period  of  infection;  they  disorganize 
rapidly  and  give  rise  to  many  coccobacilli.  At  the  beginning  of  their  formation 
the  giant  bacilli  are  slowly  motile  but  as  they  continue  to  grow  larger  motility  is 
lost.  The  normal  cells  (coccobacilli),  however,  are  very  motile.  The  cultural 
characteristics  are  as  follows:  in  ordinary  broth,  abundant  and  rapid  growth  occurs 
at  37°,  slight  sediment  after  two  days,  no  pellicle.  Upon  nutrient  agar  the  colonies 
are  yellowish  white,  large,  round,  and  somewhat  raised.  Saccate  liquefaction. 
Abundant  growth  on  serum  with  rapid  digestion  from  the  second  day.  Milk  is 
coagulated  the  third  day  at  18-20°,  no  digestion  of  casein.  The  following  carbo- 
hydrates are  fermented,  glucose,  levulose,  lactose,  saccharose,  mannit,  maltose, 
galactose,  dulcit,  and  arabinose.  Litmus  carbohydrate  media  are  more  or  less 
decolorized  with  the  exception  of  those  containing  glucose  and  saccharose. 

PATHOLOGY. — The  pathogenic  action  of  B.  lymantricola  adiposa 
manifests  itself  principally  by  the  disorganization  of  adipose  tissue. 
About  the  fifth  hour  after  inoculation  the  fat  globules  some  of  which 
are  yet  contained  in  fat  cells,  begin  to  appear  in  the  blood;  the  propor- 
tion of  these  globules  increases  rapidly  until  the  blood  becomes  milky. 
This  action  upon  adipose  tissue  is  the  first  authentic  example  of  this 
type  of  specificity  among  microbial  parasites  of  insects,  yet  this  speci- 
ficity is  only  very  relative  since  this  organism  also  multiplies  abundantly 
in  the  blood. 

PATHOGENICITY  FOR  OTHER  INSECTS. — The  larvae  of  Vanessa 
urticce,  of  the  brown  tail  moth  Euproctis  chrysorrhea,  and  the  silk 
worm  show  the  same  symptoms  upon  inoculation  as  do  the  caterpillars 
os  Lymantria  dispar. 

SAC  BROOD,  A  DISEASE  OF  BEES 
Filtrable  virus— White* 

HISTORY  AND  DISTRIBUTION. — A  disease  which  was  similar  to,  but 
was  not  foul  brood  was  noted  in  1881  by  Doolittle  in  America,  by 
Jones  of  Canada  in  1883,  and  by  Simmins  of  England  in  1887.  The 
larvae  were  found  to  die  here  and  there  throughout  the  brood  comb; 
the  disease  would  disappear  entirely  or  it  would  reappear  the  next 
season;  the  bees  would  frequently  remove  the  dead  brood  and  no 
further  trouble  would  ensue.  Simmins  found  no  microscopic  evidence 

*  White.  G.  F.     Sacbrood.     Bull.  431,  B.  of  Ent.,  U.  S.  Dept.  Agr.,  1917. 


932  MICROBIAL  DISEASES    OF   INSECTS 

of  disease  in  these  larvae.  In  1892  an  editorial  in  one  of  the  bee  jour- 
nals stated  that  dead  brood  had  been  encountered  which  did  not  seem 
to  be  infectious  and  which  lacked  two  decisive  symptoms  of  the  real 
foul  brood,  i.e.,  the  ropiness  and  the  " glue-pot"  odor.  In  1902 
G.  F.  White  of  the  U.  S.  Dept.  of  Agriculture  began  the  study  of  this 
diseased  brood.  This  disease  was  described  in  Switzerland  in  1906 
and  later  in  1910.  It  occurs  among  bees  in  localities  having  as  wide  a 
range  of  climatic  conditions  at  least,  as  are  found  in  the  United  States. 

The  name  "sac  brood"  comes  from  the  fact  that  many  larvae  dead 
of  this  disease  can  be  removed  from  the  cell  without  rupturing  their 
body  wall.  When  thus  removed  they  have  the  appearance  of  a  small 
enclosed  sac. 

SYMPTOMS. — The  strength  of  a  colony  in  which  sac  brood  is  present 
is  frequently  not  noticeably  diminished.  When  the  brood  is  badly  in- 
fected, however,  the  colony  naturally  becomes  appreciably  weakened 
thereby.  The  death  of  the  worker  larvae  is  the  primary  cause  for  the 
weakness  resulting  from  the  disease  in  a  colony.  The  colony  is  also 
weakened  by  the  dead  sacbrood  larvae  remaining  in  the  cells  for  weeks  as 
they  not  infrequently  do,  thus  reducing  the  capacity  of  the  brood  nest 
for  brood  rearing.  The  brood  dies  after  the  time  of  capping.  The  dead 
larvae  are,  therefore,  almost  always  found  extended  lengthwise  in  the  cell 
and  lying  with  the  dorsal  side  against  the  lower  wall.  It  is  not  unusual 
to  find  many  larvae  dead  of  this  disease  in  uncapped  cells.  Such  brood, 
however,  had  been  uncapped  by  the  bees  after  it  died.  In  this  disease 
the  cappings  are  frequently  punctured  by  the  bees.  Occasionally  a 
capping  has  a  hole  through  it,  indicating  that  the  capping  itself  had 
never  been  completed.  A  larva  dead  of  this  disease  loses  its  normal 
color  and  assumes  at  first  a  slightly  yellowish  tint.  "Brown"  is  the 
most  characteristic  appearance  assumed  by  the  larva  during  its  decay. 
Various  shades  are  observed.  The  term  "gray"  might  sometimes 
appropriately  be  used  to  designate  it.  The  form  of  the  larva  dead  of 
this  disease  changes  much  less  than  it  does  in  foul  brood.  The  body 
wall  is  not  easily  broken,  as  a  rule.  On  this  account,  often  the  entire 
larva  can  be  removed  from  the  cell  intact.  The  content  of  this  saclike 
larva  is  more  or  less  watery.  The  head  end  is  usually  turned  markedly 
upward.  The  dried  larva  or  scale  is  easily  removed  from  the  lower 
side  wall.  There  is  practically  no  odor  to  the  brood  combs.  Adult 
bees  are  not  susceptible  to  the  disease. 


MICROBIAL   DISEASES   OF   INSECTS  933 

CAUSAL  ORGANISM. — No  microorganisms  have  been  found  either  culturally  or 
microscopically.  However,  experimental  evidence  shows  that  the  etiological 
factor  is  a  filtrable  virus.  The  virus  contained  in  a  single  larva  recently  dead  of  the 
disease  has  been  found  sufficient  to  produce  infection  in  and  death  of  at  least  3000 
larv;e  within  a  week.  If  the  virus  from  one  larva  each  succeeding  time  were  given 
the  opportunity  of  increasing  3,ooo-fold,  in  less  than  two  weeks,  theoretically, 
a  sufficient  amount  of  virus  would  be  produced  to  infect  9,000,000  colonies,  more 
colonies  probably  than  are  to  be  found  at  present  in  the  United  States,  and  within 
three  weeks  enough  virus  could  be  produced  to  inoculate  every  colony  in  existence. 
These  figures  give  an  idea  of  the  enormous  rapidity  with  which  the  sacbrood  virus 
is  capable  of  increasing.  However,  it  is  possible  that  so  small  an  amount  of  virus 
may  be  taken  up  by  an  individual  larva  that  no  disease  results. 

The  sacbrood  virus  is  influenced  by  various  physical,  chemical  and  biological 
agencies,  which  influences,  if  applied  rightly,  may  constitute  control  measures. 
The  virus  is  killed  by  heating  in  water  at  60°  for  ten  minutes.  In  honey,  however, 
it  is  necessary  to  employ  70°  for  ten  minutes,  while  if  no  heat  is  applied  and  the  virus 
is  shielded  from  the  sun  it  remains  alive  nearly  a  month.  Drying  at  room  tempera- 
ture for  approximately  three  weeks  was  not  destructive  but  for  longer  periods  of 
time  it  was  fatal.  Dried  virus  exposed  to  the  direct  rays  of  the  sun  was  destroyed 
in  from  four  to  seven  hours;  when  suspended  in  water  from  four  to  six  hours  only  was 
required  for  destruction,  but  when  suspended  in  honey  fatal  effects  were  obtained  on 
exposure  to  sunlight  for  five  to  six  hours.  Five  days  was  required  to  destroy  the 
virus  under  the  influence  of  fermentative  processes  in  a  10  per  cent  sugar  solution  at 
room  temperature,  and  also  in  a  20  per  cent  honey  solution  at  outdoor  (summer) 
temperature.  Putrefactive  processes  (infected  larvae  crushed  and  mixed  with  soil 
in  water)  allowed  the  virus  to  remain  active  for  approximately  ten  days.  Carbolic 
acid  in  0.5,  i.o  and  2.0  per  cent  solution  was  resisted  by  the  virus  for  more  than  three 
weeks;  4.0  per  cent  is  more  effective.  However,  experiments  show  that  neither 
this  chemical  nor  quinine  should  be  relied  upon  as  a  means  of  treating  sacbrood. 

METHODS  OF  INFECTION  AND  CONTROL. — The  transmission  of  any 
brood  disease  takes  place  (i)  from  diseased  to  healthy  brood  within  a 
colony  and  (2)  from  a  diseased  colony  to  a  healthy  one.  As  has  been 
shown  experimentally  the  virus  of  sacbrood  produces  the  disease  in  all 
cases  when  larvae,  sick  and  dead  of  this  disease  are  picked  from  the 
combs,  crushed,  diluted  with  sterile  water,  the  suspension  filtered  by 
means  of  a  Berkefeld  filter  and  the  filtrate  so  obtained  fed  to  healthy 
colonies  either  directly  or  mixed  with  sirup.  From  this  fact  it  is  fair 
to  assume  that  sacbrood  may  result  whenever  the  food  or  water  used 
by  the  bees  contains  the  living  virus  of  the  disease. 

The  period  from  time  of  inoculation  to  the  appearance  of  the  first 
symptoms  of  the  disease — the  incubation  period — is  approximately 
six  days,  being  frequently  slightly  less.  By  inoculation  the  disease 


934  MICROBIAL   DISEASES    OF    INSECTS 

may  be  produced  at  any  season  of  the  year  that  brood  is  being  reared. 
Under  natural  conditions,  however,  the  disease  is  more  often  encountered 
during  the  first  half  of  the  brood-rearing  season  than  during  the  second 
half.  The  course  of  the  disease  is  not  greatly  affected  by  the  character 
or  quality  of  the  food  obtained  and  used  by  the  bees. 

Bees  have  a  tendency  to  remove  diseased  or  dead  larvae  from  the  cells. 
When  the  removal  is  attempted  about  the  time  of  death  it  is  done 
piece-meal.  Just  what  becomes  of  these  bits  of  diseased  tissue,  is  not 
known.  If  these  fragments  were  fed  to  young  healthy  larvae  within  a 
week,  they  would  most  likely  become  infected  with  sacbrood.  Exper- 
ience, however,  shows  that  under  these  conditions  the  tendency  in  a 
colony  is  in  most  cases  toward  recovery.  This  suggests  that  the 
workers  may  feed  the  infected  tissues  to  the  older  larvae  or  to  adult  bees; 
in  either  case  the  likelihood  of  the  transmission  of  the  disease  would 
apparently  be  very  materially  reduced.  If  the  infective  material 
were  stored  with  the  honey  and  did  not  reach  the  brood  in  a  month  or 
six  weeks,  again  it  is  not  probable  that  the  disease  would  be  transmitted. 

If  the  infective  material  is  removed  from  the  hive  and  freed  from 
the  adult  bees  removing  it,  experimental  evidence  indicates  that  during 
the  warmer  seasons  at  least,  there  is  but  little  chance  of  the  virus  being 
returned  to  the  hive  and  producing  any  noticeable  infection. 

There  is  also  little  probability  of  the  virus  of  sacbrood  being  trans- 
mitted by  way  of  flowers  visited  by  bees.  There  is,  however,  a  greater 
likelihood  of  the  water  supply  being  a  source  of  infection. 

Bees  drifting  or  straying  from  infected  colonies  to  healthy  ones  have 
been  proved  to  be  less  liable  to  transmit  the  disease  than  when  robbing 
occurs.  It  is  not  yet  known  in  what  way  the  sacbrood  virus  is  carried 
over  from  one  brood  rearing  season  to  another. 

From  knowledge  obtained  through  experimentation  but  few  control 
measures  can  be  advocated.  Most  of  the  advice  which  can  be  given  is 
negative.  Theoretically  it  is  better  to  store  combs  from  sacbrood  col- 
onies for  one  or  two  months  before  they  are  again  used,  as  drying  ap- 
parently destroys  the  virulence  of  the  virus  within  this  time.  After 
the  early  brood-rearing  season  of  the  year  is  past  brood  frames  from 
badly-infected  colonies  may  be  inserted  into  strong,  healthy  ones,  and 
cause  thereby  very  little  infection  and  consequently  only  a  slight  loss. 
This  is  the  preferable  method  of  disposing  of  the  infected  combs  for  the 
practical  beekeeper  to  employ.  In  practical  apiculture  no  fear  need  be 


MICROBIAL   DISEASES    OF   INSECTS  935 

entertained  that  sacbrood  will  be  transmitted  by  the  hands  or  clothing 
of  the  operator,  by  the  tools  used  about  the  apiary,  through  the  medium 
of  the  wind,  or  by  the  queen.  Flaming  or  burning  the  inside  of  the 
hive,  or  treating  the  ground  about  a  hive  containing  a  sacbrood  infected 
colony  appears  to  be  entirely  unnecessary. 

WILT  DISEASE  OR  FLACHERIE  OF  THE  GIPSY  MOTH  CATERPILLAR, 

Porthetria  dispar  L. 
Filtrable  virus — Glaser* 

HISTORY  AND  DISTRIBUTION. — There  is  no  account  of  the  occurrence 
of  wilt  in  America  prior  to  1900.  This  disease  may  have  been  intro- 
duced on  trees  or  shrubs  imported  from  Europe,  in  which  country 
"Wipfelkrankheit,"  a  wilt  disease  of  the  European  nun-moth  cater- 
pillars, Psilura  monacha,  exists.  In  Europe  flacherie  ha^  become  the 
"guardian  angel'*  of  the  central  European  forests. 

In  the  United  States  there  is  every  reason  to  suppose  that  the  wilt 
is  distributed  over  the  entire  territory  infested  by  the  gipsy  moth,  a 
territory  of  about  4,850  square  miles  (1915)  extending  over  various 
parts  of  Maine,  New  Hampshire,  Massachusetts  and  Rhode  Island. 

SYMPTOMS. — The  symptoms  of  the  wilt  disease  of  the  gipsy  moth 
caterpillars  are  those  of  flacherie  of  the  silk- worm.  They  soon  stop 
eating,  become  languid,  usually  crawl  up  on  some  object  where  they 
remain  motionless.  In  a  few  hours  there  drops  from  the  mouth  and 
anus  a  dirty,  blackish,  foul-smelling  liquid;  they  become  more  and  more 
flaccid,  one  leg  after  another  looses  its  support  and  finally  the  cater- 
pillar reduced  to  a  black  skin  is  found  hanging  limply  to  tree  trunks 
and  limbs,  still  holding  on  with  one  or  two  of  its  false  feet  or  with  the 
anal  claspers.  After  death  their  body  tissues  become  degenerated  so 
rapidly  that  it  is  impossible  to  handle  them;  a  slight  touch  breaks  the 
skin  and  a  thin  dark,  offensive-smelling  liquid  flows  out,  consequently 
they  can  never  be  used  for  histological  work. 

CAUSAL  ORGANISM. — A  filtrable  virus  seems  to  be  responsible  for  the  death  of 
these  caterpillars.  It  is  filtered  with  difficulty,  however  Bacteria  are  not  respon- 
sible for  this  disease.  Minute  dancing  granules  are  observed  in  the  Berkefeld 
filtrate,  which  may  be  etiologically  significant.  No  bacteria  or  polyhedral  bodies  are 
observed  in  the  filtrate. 

•  Glaser,  R.  W.  and  Chapman,  J.  W.  The  Wilt  Disease  of  the  Gipsy  Moth  Caterpillar. 
Jour.  Econ.  Entomol.,  6,  1913,  pp.  479-488. 

Reiff,  W.     The  Wilt  Disease,  or  Flacherie,  of  the  Gypsy  Moth,  19x1. 


936  MICROBIAL   DISEASES    OF    INSECTS 

METHODS  OF  INFECTION. — Infection  naturally  takes  place  through 
the  mouth  by  means  of  the  food.  Predisposition  to  the  disease  is  se- 
cured by  giving  the  caterpillars  food  which  has  been  placed  in  water 
and  renewed  only  every  three  or  four  days.  This  causes  an  increase  in 
the  acidity  of  the  leaves  which  in  turn  decreases  the  alkalinity  of  the 
caterpillar's  digestive  fluid.  Before  the  visible  outbreak  of  flacherie,  as 
an  early  symptom,  a  characteristic  sweet  odor  is  recognized  in  the 
breeding  cages  which  resembles  that  of  withered  lilac  blossoms  some- 
what. Whenever  this  odor  is  noticeable,  flacherie  soon  makes  its 
appearance,  and  as  it  progresses  the  odor  increases  proportionately 
(Fischer). 

Lack  of  food,  which  is  necessarily  brought  about  by  the  cater- 
pillars themselves,  causes  them  to  lose  their  vitality,  thus  producing  a 
greater  susceptibility  to  the  disease.  Defoliation  also  exposes  them 
to  the  sun's  rays  which  have  the  effect  of  converting  the  chronic  into 
the  acute  form  of  wilt.  In  lightly  infested  woodland  this  does  not  hap- 
pen as  the  caterpillars  can  always  find  shade.  Flacherie,  however, 
seems  to  be  influenced  by  climate  and  weather  conditions 
less  than  any  other  caterpillar  disease. 

Wilt  is  always  prevalent  among  the  older  caterpillars;  young  cater- 
pillars often  live  several  days  before  succumbing  to  the  disease.  Female 
caterpillars  always  succumb  more  readily  to  the  wilt  disease  than  the 
male;  this  may  perhaps  be  due  to  the  fact  that  they  require  a  longer 
time  to  mature  than  the  male.  Diseased  females  deposit  egg  clusters 
reduced  in  size,  which  contain  usually,  embryos,  incompletely  or  not 
at  all  developed.  In  this  case  there  are  always  found  undeposited 
eggs  within  the  body  of  the  female,  which  never  occurs  with  healthy 
moths.  Genetic  immunity  of  certain  individuals  is  probable.  Sub- 
lethal  doses  of  the  virulent  nitrate  may  produce  active  immunization. 
Although  probable,  there  is  yet  no  definite  evidence  that  wilt  is  trans- 
mitted from  one  generation  to  another. 

PATHOLOGY  OF  WILT. — When  a  caterpillar  dies  of  wilt,  all  of  its 
tissues  are  in  a  state  of  disintegration.  The  intestine  is  the  last  in- 
ternal organ  to  disintegrate.  A  smear  of  the  brown  liquid  from  a  dead 
caterpillar  examined  microscopically  with  a  high  power  lens  will  be 
found  to  contain,  besides  the  elements  of  disorganized  tissues,  myriads 
of  highly  refractive  polyhedral  bodies  of  various  sizes.  The  average 
polyhedron  measures  from  I/A  to  6ju  in  diameter  and  is  never  regular  as 


MICROBIAL   DISEASES    OF   INSECTS  937 

are  the  silk-worm  polyhedra.  The  significance  of  these  bodies  is  not 
known.  However,  they  are  believed  to  be  reaction  bodies  belonging  to 
the  highly  differentiated  albumins,  the  nucleoproteids.  They  may  be 
stages  of  the  filtrable  virus  but  no  evidence  has  been  brought  forward 
to  substantiate  this  view.  It  has  been  determined  however,  that  no 
diagnosis  of  wilt  is  valid  unless  polyhedra  are  demonstrated 
microscopically. 

The  "Wipfelkrankheit"  of  the  nun-moth  in  Germany  is  essentially 
the  same  disease  as  that  of  the  gipsy  moth  in  the  United  States  (Es- 
cherich  and  Miyajima.) 

PEBRINE,  AN  INFECTIOUS  DISEASE  OF  THE  SILK- WORM,  Bombyx 

mori 

Nosema  bombycis 

HISTORY  AND  DISTRIBUTION. — About  the  year  1853,  anxious  atten- 
tion began  to  be  given  in  the  southern  part  of  France  to  the  ravages  of 
a  disease  among  silk- worms  which  from  its  alarming  progress,threatened 
to  issue  in  national  disaster.  Symptoms  of  this  disease  had  been  noted 
as  early  as  1845.  It  finally  became  necessary  to  import  seed  (technical 
term  for  eggs)  for  continuing  the  culture  of  the  silk-worms.  This  was 
procured  first  from  Lombardy,  but  after  one  successful  year  the  same 
disappointments  occurred.  Then  Italy  was  attacked,  also  Spain  and 
Austria;  later  seed  was  procured  from  Greece,  Turkey,  the  Caucasus, 
but  to  no  avail;  China  itself  was  attacked  and  in  1864,  healthy  seed 
could  be  obtained  only  from  Japan. 

This  disease,  characterized  by  dark  spots  on  the  silk-worms,  was 
called  pebrine,  from  the  patois  word  pebre  (pepper),  the  name  given  to 
it  by  de  Quatrefages  on  account  of  the  resemblance  of  these  spots  to 
pepper  grains. 

SYMPTOMS. — As  above  mentioned,  one  of  the  symptoms  of  pebrine 
is  the  manifestation  of  dark  spots  in  the  skin  of  the  larvae;  some  worms 
languish  on  the  frames  in  their  earliest  days,  others  in  the  second 
stage  only,  some  pass  through  the  third  and  fourth  molts,  climb  the 
twig  and  spin  their  cocoons.  The  chrysalis  becomes  a  moth,  but  the 
moth  shows  signs  of  disease  in  its  deformed  antennae  and  withered  legs; 
the  wings  seem  singed.  Eggs  from  these  moths  are  inevitably  un- 
successful the  following  year.  Thus,  in  the  same  nursery  in  the  course 


938  MICROBIAL  DISEASES    OF   INSECTS 

of  the  two  months  that  it  takes  a  larva  to  become  a  moth,  the  pebrine 
disease  is  alternately  sudden  or  insidious;  it  bursts  out  or  disappears, 
it  hides  itself  within  the  chrysalis  and  reappears  in  the  moth  or  the  eggs 
of  a  moth  which  has  seemed  sound. 

CAUSAL  ORGANISM. — The  causal  organism  for  this  disease  is  Nosema  bombycis, 
a  protozoon  belonging  to  the  Microsporidia.  The  spores  find  their  way  from  the 
caterpillar  by  means  of  the  dejecta  or  through  the  disintegration  of  dead  forms  to 
other  silk  worms.  Some  of  the  parasites  find  their  way  into  the  ovary,  produce 
spores,  pass  through  the  pupal  and  imaginal  stages  of  the  host  into  the  next  genera- 
tion of  silk-worms.  The  spores  are  often  regarded  as  pe"brine.  corpuscles. 

In  the  worms  suffering  from  pebrine,  corpuscles  or  polyhedral  bodies,  first  noted 
by  Pasteur,  are  found  in  all  tissues  and  all  fluids  of  the  body,  even  in  the  material 
from  which  the  silk  is  made;  naturally  they  are  also  found  in  the  dejecta  of  the  worms. 
These  same  bodies  are  found  in  and  on  the  infected  eggs,  pupae  and  moths  and  in 
innumerable  quantities  in  the  dust  of  the  infected  nurseries;  they  are  easily  rec- 
ognized microscopically. 

These  polyhedral  bodies  are  now  known  not  to  be  the  etiological  factor,  but  are 
most  probably  an  effect  of  the  disease.  Glaser  and  Chapman*  have- found  them  to 
be  nucleo-protein  crystal-like  degeneration  products  of  the  insect  blood  cells,  and 
not  organisms.  They  contain  iron  and  phosphorus.  Crystals  simulating  the 
original  polyhedra  are  obtained  on  dissolving  polyhedra  in  alkali  dialyzing  out  the 
alkali,  and  evaporating  the  protein  solution.  Before  their  nature  was  known,  how- 
ever, elimination  of  all  eggs  containing  these  bodies  resulted  in  the  suppression  of 
the  disease. 

METHODS  or  INFECTION. — A  very  common  method  of  infection  is 
due  to  the  habits  of  the  silk-worms  crawling  over  one  another.  When 
a  worm  moves  across  a  diseased  worm,  its  claws  cut  through  the  tegu- 
ment and  become  contaminated;  in  its  progress  it  inoculates  other  worms 
by  means  of  its  soiled  fangs.  The  greatest  source  of  contagion,  however, 
is  the  excreta  which  fall  on  the  food  of  the  worms.  Luckily  this  infec- 
tious material  on  being  exposed  to  light  and  air,  becomes  rapidly  at- 
tenuated. However,  the  causal  organism  is  not  so  attenuated  when 
within  the  egg;  it  passes  the  winter  in  a  latent  state  and  develops  along 
with  the  worm,  multiplying  within  its  body  and  altering  more  or  less 
profoundly  the  conditions  of  existence. 

CONTROL. — If  moths  are  not  seriously  diseased,  their  eggs  will 
always  furnish  several  healthy  larvae  and  if  these  are  isolated  as  soon 
as  they  hatch  out  and  are  kept  and  bred  under  sanitary  conditions,  a 

*Glaser,  R.  W.  and  Chapman,  J.  W.  The  nature  of  the  polyhedral  bodies  found 
in  insects.  Biol.  Bui.  Marine  Biol.  Lab.  Woods  Hole,  Mass.,  30,  pp.  367-390, 1916. 


MICROBIAL   DISEASES    OF    INSECTS  939 

race  of  worms  free  from  corpuscles  can  soon  be  obtained.  This  has 
been  found  to  be  the  most  effective  method  of  combating  pebrine 
Excessive  heat  saps  the  vitality  of  the  silk-worm  and  makes  it  ready 
prey  to  disease.  Open  air  cages  result  in  much  hardier,  more  active 
worms. 

NOSEMA-DISEASE  OF  BEES 

Nosema  apis — Zander 

HISTORY  AND  DISTRIBUTION. — This  disease  was  apparently  first 
noticed  about  sixty  years  ago  by  Donhoff  (1857)  who  discovered, 
microscopically,  small  oval  bodies  in  the  stomachs  of  adult  bees  which 
he  supposed  had  died  of  exposure.  He  sent  some  of  the  bees  to  Leuck- 
hart  who  after  examining  them  microscopically  expressed  the  belief 
that  the  oval  bodies  were  the  spores  of  a  fungus.  So  Donhoff  referred 
to  this  bee  disorder  as  "Pilzsucht"  (fungous  disease).  In  1909  Zander 
found  similar  small  oval  bodies  in  the  walls  of  stomachs  taken  from 
affected  bees  and  to  these  organisms  he  gave  the  name,  "Nosema  apis" 
and  for  the  disease  he  (1911)  used  the  name  "Nosemaseuche."  This 
disease  is  known  in  Europe  under  similar  names,  "  Nosemakrankheit" 
in  Switzerland  (Nussbaumer,  1912;  Angst,  1913),  "Nosemasygdommen" 
in  Denmark  (Bahr,  1915).  Nosema  infection  has  been  reported  also 
from  Australia  (1910),  Brazil  (1911),  Canada  (1914),  England  (1911), 
and  Germany  (1909).  White*  has  found  Nosema-disease  in  samples 
of  bees  received  from  27  different  states  of  the  United  States.  The 
distribution  in  the  United  States,  however,  has  been  hard  to  determine 
as  beekeepers  have  not  learned  to  recognize  the  disease  produced  by 
Nosema  apis  by  any  one  name.  " Spring  dwindling"  and  "weakened 
colonies"  were  descriptive  terms  applied  by  beekeepers  to  colonies  in 
which  Nosema  apis  was  found.  The  highest  percentage  of  Nosema- 
infected  bees  occurred  in  weak  colonies. 

SYMPTOMS. — Nosema-disease  is  an  infectious  disease  of  adult  honey- 
bees. It  is  not  particularly  malignant  in  character,  being  more  in  this 
respect  like  sacbrood  than  the  foulbroods.  So  far  as  is  known,  all  races 
of  honey-bees  are  susceptible.  This  disease  presents  only  a  few  symp- 
toms. These  White  described  as  characteristic  of  the  colony  rather 
than  of  the  individual  as  a  unit,  since  it  is  the  colony  as  a  whole  that  is 
of  primary  interest  to  beekeepers. 

"•"White,  G.  F.     Nosema-disease,  Bui.  780,  U.  S.  D.  A.  1919,  pp.  59. 


940  MICROBIAL   DISEASES    OF    INSECTS 

Weakness  is  a  colony  symptom  which  will  invariably  be  manifest 
if  a  sufficiently  large  percentage  of  the  bees  of  the  colony  are  Nosema- 
infected  and  if  the  infection  persists  for  a  sufficient  period.  When  only 
a  small  percentage  of  the  bees  are  infected  the  weakness  resulting  may 
never  be  apparent.  The  loss  in  strength  may  be  gradual  or  sudden. 

A  Nosema-infected  colony  behaves  similarly  to  a  healthy  one. 
The  stores  are  sufficient.  The  queen  does  her  work  well.  As  the 
colony  dwindles  the  queen  is  usually  among  the  last  handful  of  bees. 
The  brood  in  general  is  normal  in  appearance,  but  in  colonies  weakened 
by  the  disease  not  infrequently  it  is  seemingly  in  excess  of  the  amount 
that  can  be  properly  cared  for  by  the  adult  bees  present.  The  workers, 
especially  the  young  ones,  are  most  frequently  infected,  although  drones 
and  queens  are  susceptible.  It  is  not  unusual  to  find  from  ten  to  twenty 
per  cent  of  the  workers  of  diseased  colonies  infected.  An  infected, 
bee  manifests  no  outward  symptoms  of  the  disease  when  seen  among  the 
other  bees  of  the  colony  and  it  performs  functions  similar  to  those  per- 
formed by  healthy  ones. 

When  the  stomach  of  an  infected  bee  is  removed  it  may  show  marked 
changes  which  are  characteristic  of  Nosema-disease.  The  brownish 
yellow  or  dark  reddish  hue  of  the  normal  stomach  becomes  gradually 
lost  as  the  disease  advances.  The  organ  is  often  increased  in  size, 
the  constrictions  are  less  marked,  and  the  transparency  is  diminished. 
In  late  stages  of  the  disease,  however,  the  stomach  approaches  the 
normal  in  size  and  the  constrictions  are  again  well  marked.  The  organ 
is  then  white  and  opaque  and  the  tissues  are  fraible  and  easily  crushed. 
When  crushed  the  mass  presents  a  milky  appearance. 

Upon  microscopic  examination,  Nosema  apis  is  found  in  very  large 
numbers  in  the  crushed  tissues.  The  presence  of  the  parasite  is  almost 
invariably  recognized  by  its  spore  form.  The  presence  of  Nosema- 
infected  bees  in  a  colony  is  the  one  constant  colony  symptom  of  the 
disease: 

CAUSAL  ORGANISM. — White  proves  that  Nosema  apis  is  the  cause  of  Nosema- 
disease  by  a  process  of  elimination.  Maiden  (1912-1913)  who  studied  the  bacter- 
iology of  Nosema-infected  bees  found  that  although  the  number  of  bacteria  in  dis- 
eased bees,  was  much  greater  than  in  normal  ones,  there  was  no  evidence  of  a  direct 
etiological  relation  existing  between  these  bacteria  and  the  disease.  White  himself 
found  that  Nosema-disease  is  not  caused  by  a  filtrable  virus.  Higher  animal 
parasites  and  fungi  being  absent,  and  the  bacteria  and  the  filtrable  viruses  thus  being 
eliminated,  tentatively  at  least,  there  remains  only  one  group,  the  protozoa,  and  of 


MICROBIAL  DISEASES   OF   INSECTS  941 

this  group  there  is  only  one  species,  Nosema  apis,  that  is  constantly  present  in 
Nosema-disease.  The  conclusion  is  naturally  reached,  therefore,  that  Nosema  apis 
is  the  cause  of  Nosema-disease.  Such  a  conclusion  is  in  harmony  with  views 
generally  accepted  at  the  present  time  in  regard  to  proof  necessary  to  establish  the 
causal  relation  of  such  a  germ  to  the  disease. 

The  spore  form  of  Nosema  apis  is  the  form  most  encountered  and  most  readily 
recognized  in  making  an  examination  for  the  parasite.  These  spores  in  unstained 
(India  ink)  preparations  are  small,  refractile,  more  or  less  oval  bodies  varying  some- 
what in  size,  but  having  an  average  length  of  4.46/1  and  an  average  breadth  of  2.44/1. 
In  stained  preparations  the  average  length  and  breadth  are  4.15/1  and  2.06/1  respec- 
tively. The  spore  is  surrounded  by  a  somewhat  resistant  coat  which  tends  to 
maintain  for  it  a  constant  form,  but  it  is  not  a  rigid  structure,  since,  when  studied  in 
fresh  preparations  it  will  be  seen  to  bend  to  and  fro  as  it  is  carried  along  by  a  current 
under  the  coverglass.  A  source  of  confusion  in  the  diagnosis  of  Nosema  apis  in 
adult  bees  is  the  fact  that  starch  granules  from  pollen  grains  of  corn  and  of  most  of 
the  cereals  closely  resemble  the  spores  of  Nosema  apis.  This  organism  has  not 
been  cultivated  in  pure  culture  by  artificial  methods. 

Nosema  apis  suspended  in  water  is  destroyed  by  heating  for  ten  minutes  at 
58°;  when  suspended  in  honey,  59°  is  required  for  destruction.  When  dried  at  room 
and  outdoor  temperatures  respectively  it  remained  virulent  for  about  two  months, 
at  incubator  temperature  about  three  weeks,  and  in  a  refrigerator  about  seven  and 
one-half  months.  When  dry,  fifteen  to  thirty-two  hours  direct  exposure  to  the 
sun's  rays  is  necessary  for  total  destruction;  when  suspended  in  water  thirty-seven 
to  fifty-one  hours  is  required,  while  if  suspended  in  honey,  destruction  is  frequent  as 
the  temperature  of  the  honey  reaches  or  exceeds  60°,  a  temperature  at  which  the  germ 
is  killed  by  heat.  If  placed  in  honey  and  shielded  from  the  light  Nosema  apis 
remains  virulent  for  two  to  four  months  at  room  temperature.  Fermentative 
processes  are  destructive  to  the  spores  in  a  20  per  cent  honey  solution  in  three  days 
at  incubator  temperature,  and  in  nine  days  at  outdoor  temperature  while  in  a  10 
per  cent  sugar  solution  it  is  destroyed  in  from  seven  to  eleven  days  at  room  tempera- 
ture. When  subjected  to  putrefactive  processes  (suspensions  made  in  a  i  per  cent 
aqueous  peptone  solution)  Nosema  apis  resists  destruction  for  five  days  at  incubator 
temperature,  for  two  weeks  at  room  temperature,  and  for  more  than  three  weeks  at 
outdoor  temperature.  Nosema  apis  spores  in  the  bodies  of  dead  bees  cease  to  be 
virulent  in  one  week  at  incubator  temperature,  in  four  weeks  at  room  temperature, 
in  six  weeks  at  outdoor  temperature  and  in  four  months  in  a  refrigerator,  while  if 
the  bodies  of  the  dead  bees  are  lying  on  the  soil  virulence  ceases  in  from  forty-four 
to  seventy-one  days.  A  i  per  cent  solution  of  carbolic  acid  destroys  the  spores  of 
this  organism  in  less  than  ten  minutes. 

METHODS  or  INFECTION. — In  general,  the  manner  in  which  a  bee 
becomes  infected  with  Nosema  apis  is  as  follows:  Spores  which  have  left 
the  body  of  an  infected  bee  with  the  excrement  are  ingested  by  the  healthy 
adult  bee.  As  the  excrement  is  usually  voided  in  flight  this  influences 
the  chance  of  infection.  The  environment  within  the  stomach  of 


942  MICROBIAL  DISEASES    OF   INSECTS 

the  bee  is  favorable  for  the  growth  and  multiplication  of  the  parasite. 
The  digestive  fluids  are  believed  to  assist  in  removing  the  spore  coat. 
The  liberated  young  parasite  finds  its  way  to  the  walls  of  the  stomach 
and  invades  the  epithelial  cells.  Within  this  epithelial  tissue  it  grows 
and  multiplies  with  great  rapidity,  giving  rise  finally  to  numerous 
spores.  The  cells  of  the  epithelium  at  times  seem  to  become  virtually 
filled  with  the  parasites.  That  portion  of  an  epithelial  cell  that  is 
normally  shed  into  the  lumen  of  the  stomach  in  case  of  infection  bears 
with  it  many  spores.  These  are  liberated  gradually  from  the  frag- 
ments, become  mixed  with  the  partially  digested  food  in  the  stomach, 
and  are  carried  onward  first  into  the  small  and  then  into  the  large 
intestines,  and  finally  pass  out  of  the  alimentary  tract  with  the  excre- 
ment. Other  bees  ingesting  these  spores  become  infected.  This,  in 
brief,  is  the  life  cycle  through  which  the  parasite  passes. 

In  infecting  the  stomach  the  -parasite  reaches  the  basement  mem- 
brane but  does  not  penetrate  it.  The  muscular  part  of  the  organ  is, 
therefore,  uninvolved.  Likewise  when  found  in  the  Malpighian 
tubules  the  infection  does  not  proceed  beyond  the  basement  membrane. 
The  protozoon  does  not  infect  the  pharynx,  the  oesophagus,  the  honey 
sac,  the  proventriculus,  the  small  or  the  large  intestine — organs  which 
possess  a  pronounced  chitinized  intima.  So  far,  Nosema  apis  has  not 
been  encountered  in  the  blood,  musculature  or  any  of  the  other  tis- 
sues of  the  body. 

Infection  in  apiaries  has  been  found  to  occur  at  all  seasons  of  the 
year  but  is  greatest  during  the  spring.  Experimentally,  however,  it 
has  been  found  that  bees  are  susceptible  to  Nosema  apis  infection  the 
year  round.  The  role  played  by  food  in  the  causation  of  Nosema- 
disease  is  slight,  if  indeed  it  contributes  at  all  appreciably  to  it. 

The  fact  determined  experimentally,  that  a  suspension  of  Nosema 
apis  in  syrup  when  fed  to  bees  will  produce  the  disease,  shows  quite 
conclusively,  however,  that  infection  takes  place  through  the  ingestion 
of  the  parasite.  At  present  there  is  no  evidence  that  it  takes  place 
otherwise  than  by  way  of  the  alimentary  tract.  The  facts  which  are 
known  concerning  Nosema-disease  indicate  that  the  disease  may  be 
transmitted:  (i)  From  the  infected  bees  of  a  colony  to  healthy  bees 
of  the  same  colony,  and  (2)  from  the  infected  bees  of  a  colony  to  heal  thy 
bees  of  another  colony.  Under  certain  circumstances  the  infection  is 
not  readily  transmitted  within  the  hive.  For  example,  colonies  which 


MICROBIAL  DISEASES   OF   INSECTS  943 

in  the  spring  of  the  year  show  less  than  50  per  cent  of  Nosema-infected 
bees  are  likely  to  recover  from  the  infection  without  treatment.  A 
colony  may  contain  a  small  percentage  of  Nosema-infected  bees 
throughout  the  year  and  not  become  heavily  infected  at  any  time. 
Colonies  experimentally  inoculated  in  June,  July,  or  August  are  practi- 
cally free  from  the  infection  within  six  weeks.  This  is  probably  due 
to  the  fact  that  the  young  bees  replace  those  dying  of  the  infection. 

To  the  contrary,  however,  when  heavy  losses  occur  among  the 
workers  in  the  spring  the  colony  suffers  as  there  are  no  young  bees  to 
replace  those  lost  as  a  result  of  the  infection. 

Colonies  which  die  out  or  become  weakened  by  the  disease  furnish 
.conditions  which  invite  robbing.  Robbing  in  a  certain  number  of 
cases  probably  results  in  the  transmission  of  the  disease  but  this  like- 
lihood seems  not  to  be  nearly  as  great  with  Nosema-disease  as  in  the 
case  of  the  foulbroods. 

Death  from  Nosema-infection  takes  place  during  the  active  bee 
season  in  from  two  weeks  to  a  month;  during  winter  the  disease  may  run 
two  or  three  months  or  even  more.  Infected  drones  die  sooner  than 
infected  workers  but  infected  queens  probably  live  longer.  It  is  quite 
likely  that  the  age  of  the  bee  when  infected  is  not  a  negligible  factor  in 
determining  the  course  of  the  disease.  Whether  a  bee  once  infected 
ever  recovers  from  the  infection  has  not  yet  been  established  definitely. 
From  what  is  known  of  diseases  in  man  and  animals,  however,  recovery 
might  be  expected  in  a  certain  percentage  of  Nosema-infected  bees. 
The  data  so  far  indicate  that  recovery  from  Nosema-infection  among 
worker  bees  is  comparatively  rare. 

METHOD  or  CONTROL. — Experiments  show  that  brood  combs  need 
not  be  destroyed  as  no  Nosema-infection  occurs  even  when  brood-combs 
from  Nosema-infected  colonies  are  inserted  immediately  into  healthy 
colonies.  When  medicated  diluted  honey  is  fed  to  Nosema-infected 
colonies,  using  different  drugs,  some  drugs  prove  efficaceous  while 
others  have  no  effect.  White  states  that  these  latter  experiments  are 
altogether  too  few  for  definite  conclusions  as  to  the  extent  of  their  action. 

Two  probable  sources  of  infection  are  the  presence  of  a  sluggish 
body  of  water  near  an  apiary  which  is  used  by  bees  as  a  water  supply 
and  the  robbing  of  diseased  colonies,  and  these  are  more  or  less  under 
control.  The  disease  does  not  seem  to  be  spread  through  the  medium 
of  flowers,  by  the  hands  and  clothing  of  the  apiarist,  the  tools  used 


944  MICROBIAL   DISEASES    OF    INSECTS 

about  an  apiary,  winds,  hives  which  have  housed  infected  colonies, 
combs  from  such  colonies,  or  bees  dead  of  the  disease  unless  they  serve 
to  contaminate  the  water  supply.  Thus  these  latter  mentioned  im- 
probabilities can  be  eliminated  when  dealing  with  control  measures. 

MISCELLANEOUS  INSECT  DISEASES 
ENTOMOPHTHORACE^E  * 

ENTOMOGENOUS  FUNGI.  * — Empusa  musctz  is  the  best  known  mem- 
ber of  this  group  of  insect  parasites.  This  species  attacks  the  house 
fly.  The  fly  weakened  by  the  disease  fastens  itself  to  the  wall  or  win- 
dow pane  by  its  mouth  parts.  The  mold  fruiting  upon  the  dead  body 
discharges  its  spore  masses  which  adhere  where  they  strike.  Such 
dead  flies  are  frequently  surrounded  by  a  discolored  circle  where  great 
numbers  of  these  spores  are  fixed  upon  the  wall.  Natural  epidemics 
of  this  kind  frequently  destroy  flies  and  other  insects  but  attempts  to 
produce  such  epidemics  by  inoculation  have  generally  failed  because 
the  fungus  is  too  greatly  dependent  upon  uncontrollable  factors  of 
temperature  and  humidity. 

Other  entomogenous  fungi  include  representatives  of  many  different 
groups.  The  life  histories  of  some  of  them  are  well-known  but  many 
of  them  have  been  only  partially  studied.  The  practical  usefulness 
of  some  of  these  species,  notably  Sporotrichum  globuliferum,  as  a  chinch- 
bug  disease,  has  been  studied  carefully.  While  the  work  was  markedly 
successful  in  causing  an  epidemic  disease  when  conditions  favored  it, 
dependence  upon  particular  conditions  was  so  complete  that  the 
production  of  the  disease  as  an  effective  destroyer  of  pests  failed. 
Similar  results  have  attended  the  effort  to  use  other  fungi  as  insect- 
destroyers.  The  conditions  which  make  possible  their  development  in 
epidemic  form  only  occur  occasionally.  These  conditions  in  themselves 
are,  as  a  rule,  very  unfavorable  to  insects.  Under  other  climatic  con- 
ditions, these  diseases  appear  only  as  isolated  cases,  negligible  in  their 
effect  upon  the  insect  population,  no  matter  how  carefully  the  inoculat- 
ing material  is  spread  by  man. 

One  great  series  of  fungi  the  Laboulbeniales  is  limited  to  insect 
parasitism.  The  genera  and  species  of  this  group  are  very  specialized 

*  Prepared  by  Charles  Thorn. 


MICROBIAL   DISEASES    OF   INSECTS  945 

forms  producing  local  lesions  and  typical  fruit  bodies.     Thaxter  has 
described  and  figured  a  great  number  of  these  species. 

OTHER  MICROBIAL  DISEASES* 

A  new  saccharomycete,  Monosporella  unicuspidata,  n.g.  and  n.sp., 
was  found  by  Keilin  to  be  parasitic  in  the  body  cavity  of  the  ceratopog- 
noid  larva  (Dasyhelea  obscura)  which  usually  lives  in  the  thick  brown 
sap  that  fills  the  infected  wounds  of  elm  or  horse  chestnut. 

Paillot  describes  five  species  of  coccobacilli  which  infect  the  cater- 
pillar of  Pieris  brassica,  namely,  B.  pieris  fluorescens,  B.  pieris  lique- 
faciens,  B.  pieris  non-liquefaciens  a  and  B.  non-liquefaciens  ft  and  B. 
pieris  agilis. 

Three  new  bacteria  parasitic  in  the  caterpillar  of  the  gipsy-moth 
have  also  been  described  by  Paillot.  One  of  these  resembles  B.  lyman- 
tria  of  Picard  and  Blanc,  the  other  two  are  Diplococcus  lymantria,  a 
species  quite  different  from  that  of  Hanneton,  which  causes  a  very 
energetic  phagocytosis  to  take  place  in  the  blood  of  the  insect  but  is 
not  very  pathogenic,  and  Bacillus  liparis  which  resembles  Bad. 
diphtheria  morphologically.  This  last  named  organism  also  induces 
a  very  active  phagocytosis,  but  is  more  pathogenic  than  D.  lymantrice. 

A  silk  worm  disease  in  Japan  is  produced  by  a  spore-forming  organ- 
ism Bacillus  sotto  ("Sotto"  is  a  Japanese  term  signifying  "sudden 
decay  "),  the  ill  effects  of  which  are  due  to  a  toxin  which  largely  remains 
fixed  in  the  organism  and  in  the  spores.  Atoxogenic  strains  of  this 
bacillus  occur  which  can  be  distinguished  from  toxogenic  strains  neither 
culturally  nor  through  immunization.  (Aoki  and  Chigasaki). 

Paillot  has  found  the  cockchafer  to  be  susceptible  to  many  varieties 
and  races  of  coccobacilli  which  cause  septicemias.  Four  types  of 
B.  melolontha  are  found,  one  a  liquefying  type,  the  other  three  non- 
liquefying.  In  about  30  per  cent  of  the  septicemias  caused  by  B. 
melolontha  a  secondary  infection  occurs.  Three  different  associated 
diseases  have  been  studied,  due  (i)  to  B.  melolonthce  nonliquefaciens  ft 
and  a  Gram-positive  diplococcus,  Diplococcus  melolontha,  (2)  to  B. 
melolontha  liquefaciens  and  Diplococcus  melolonthae,  and  (3)  to  B. 
melolontha  liquefaciens  and  a  large  sporulating  bacillus,  B.  hoplo- 
sternus.  B.  hoplosternus  is  very  pathogenic  for  the  cockchafer  and  the 
caterpillars  of  Vanessa  urtica,  Euproctis  chrysorrhea,  Chelonia  caja, 

*  Prepared  by  Z.  Northrup  Wyant. 
60 


946  MICROBIAL  DISEASES    OF   INSECTS 

Malacosoma  neustria,  Hanneton  commun  and  Hanneton  de  la  Saint- Jean 
(Rhyzotrogus  solsticialis) ,  but  does  not  kill  the  gipsy  moth  caterpillar, 
Lymantria  dispar,  regularly,  even  after  many  passages. 

Serbinov  describes  an  infectious  diarrhoea  of  bees  on  the  south  coast 
of  the  gulf  of  Finland  due  to  two  new  bacteria,  Bad.  coli  apium  n.sp. 
and  Proteus  alveicola  n.  sp.  The  disease  develops  rapidly  and  becomes 
epizootic,  the  bees  become  weakened  and  death  follows,  frequently  in 
convulsions. 

Korke  of  India  describes  a  new  protozoon,  Nosema  pulicis  n.  sp. 
which  has  been  found  to  cause  frequent  infections  of  the  digestive  tract 
of  the  dog  flea,  Ctenocephalus  felis.  This  parasite  was  infected  so 
readily  that  in  about  three  weeks  the  infection  rose  from  about  16  per 
cent  to  nearly  100  per  cent  under  controlled  conditions. 

Bacillus  erausquinii  n.  sp.  was  isolated  from  locusts  of  the  species 
Romalea  miles  in  Argentina  by  Cullen  and  Maggio.  It  is  said  to  have 
many  characteristics  which  distinguish  it  from  B.  acridiorum. 

A  disease  of  the  caterpillars  of  Gortyna  ochracea,  an  artichoke  pest, 
is  recorded  in  the  Department  of  Var,  France.  Bacillus  gortyna  was 
isolated  as  the  causal  factor. 

Bacillus  pyrameis  I  and  II  were  isolated  from  the  blood  and  tissues 
of  the  caterpillars  of  Pyrameis  cardui,  another  artichoke  pest.  These 
may  be  distinct  or  merely  varieties  of  a  single  species;  they  may  repre- 
sent one  or  more  saprophytic  species  widespread  in  nature  which  are 
readily  adaptable  to  a  parasitic  life  (Paillot). 

Two  associated  microorganisms,  one  a  motile  rod  and  the  other  a 
coccus  were  the  cause  of  epizootics  destroying  nearly  all  of  the  cater- 
pillars of  Galleria  melonella,  the  bee  moth,  which  were  being  raised  for 
experimental  purposes  (Metamikov).  The  rod  form  was  the  more  viru- 
lent on  injection.  The  manner  in  which  infection  takes  place  was  not 
determined. 

GENERAL  PATHOLOGY  AND  IMMUNITY  STUDIES 

The  study  of  degenerative  changes  in  normal  and  in  pathological 
blood  cells  of  insects  is  especially  important  as  the  blood  is  frequently 
used  in  diagnosing  the  health  of  a  particular  insect.  Glaser*  (1917) 

*  Glaser,  R.  W     The  growth  of  insect  blood  cells  in  vitro.    Psyche  24,  1917- 
pp.  1-6,  i  pi. 


MICROBIAL   DISEASES    OF    INSECTS  947 

succeeded  not  only  in  growing  normal  insect  blood  cells  in  vitro  but  in 
observing  in  vitro  the  formation  of  typical  polyhedra  from  the  blood  of 
healthy  M .  americanum  and  Porthetria  dispar  larvae  fed  with  filtered 
polyhedral  virus  (Berkefeld  "N"  filter  used).  He  found  that  it  was 
impossible  to  infect  the  blood  directly  with  the  virus.  The  virus  had 
to  be  given  "a  start"  within  the  insect  itself.  Later  stages  of  the  virus 
however,  find  the  conditions  suitable  on  the  tissue  culture  slides. 

Glaser  observed  from  his  studies  that  normal  blood  cells  have  a 
normal  tendency  towards  crystalline  disintegration,  thus  it  is  not  sur- 
prising that  crystals  (polyhedra)  are  found  within  the  degenerating 
nuclei  in  a  series  of  insect  diseases. 

The  ability  of  insect  tissue  to  grow  well  seems  to  vary  to  a  slight 
degree  according  to  the  species  of  the  insect. 

Glaser  (1918)  in  studying  immunity  principles  in  insects  found  that 
while  entomological  text-books  emphasize  the  importance  of  phago- 
cytosis by  blood  cells  called  amebocytes,  in  ridding  the  insect  body  of 
foreign  matter,  in  reality  these  insect  blood  cells  are  visibly  rather 
passive.  Experiments  on  various  insects  with  bacteria  pathogenic 
for  the  species,  showed  that  the  normal  blood  did  act  antagonistically 
toward  the  organisms  introduced,  but  the  riddance  was  not  accom- 
plished by  hungry  amebocytes;  the  only  movement  shown  by  these 
blood  cells  in  vitro  consisted  in  cell  division.  The  antagonistic  sub- 
stances are  extracellular  and,  therefore,  in  the  blood  plasma  or  serum. 
Microscopic  observation  with  grasshopper,  army  worm  and  gipsy 
moth  caterpillar  blood  showed  that  when  apparent  phagocytosis  oc- 
curred the  infecting  bacteria  seemed  to  bore  their  way  into  the  cyto- 
plasm. This  may  have  been  due  to  surface  tension,  however,  and 
might  be  called  phagocytosis  if  the  word  is  used  in  a  broad  sense.  On 
the  culture  slides,  the  quantity  of  the  blood  is  not  sufficient  and  meta- 
bolism is  lowered,  so  that  antagonistic  substances  are  not  formed  so 
rapidly  nor  so  abundantly  as  is  the  case  within  the  body  of  the  insect. 

The  blood  of  several  grasshoppers  (Melanopltis  femur-rubrum) 
which  had  been  immunized  to  Bacillus  poncei  by  the  injection  of  o.i  cc. 
of  a  twenty- four  hour  broth  culture  was  successfully  used  in  demon- 
strating the  presence  of  a  specific  agglutinin.  The  blood  of  uninfected 
grasshoppers  failed  to  show  this  phenomenon.  The  presence  of  bac- 
tericidal substances  in  immune  insect  serum  was  also  proven.  Actively 
immunized  grasshopper  blood  showed  a  high  degree  of  antagonism 


948  MICROBIAL  DISEASES    OF   INSECTS 

toward  the  bacteria  used  in  producing  this  immunity.  The  extent  of 
immunity  studies  possible  with  insects  seems  to  be  limited  by  the  fact 
that  the  insects,  grasshoppers,  and  caterpillars,  do  not  seem  to  be  able 
to  overcome  the  effects  of  a  second  injection. 

The  agglutination  reaction  has  been  successfully  applied  by  Aoki 
and  Chigasaki  in  the  examination  of  silkworms  for  differentiating  B. 
sotto  from  B.  megaterium  and  B.  alvei  which  it  resembles,  as  this  re- 
action is  strongly  specific  for  the  former.  Aoki  has  also  worked  with 
the  precipitation  reaction  using  silk  worm  caterpillar  immune  serum. 


DIVISION  X* 
MICROBIAL  DISEASES  OF  PLANTS 

INTRODUCTION 

Although  the  earliest  study  of  bacterial  diseases  in  plants  antedates 
the  isolation  of  the  tubercle  bacterium  and  the  cholera  spirillum,  this 
branch  of  bacteriology  has  not  been  marked  by  the  progress  which  has 
characterized  the  investigation  of  animal  diseases.  The  loss  of  a  human 
life  or  of  a  valuable  domestic  animal  has  appealed  to  the  student  of 
disease  more  strongly  than  the  blighting  of  a  pear  tree,  or  the  wilting  of 
a  potato  vine,  and,  quite  naturally,  he  has  directed  his  efforts  along  those 
lines  which  have  offered  the  greater  inducements,  and  which  have 
demanded  immediate  attention. 

However,  with  the  introduction  of  new  plants,  foreign  seeds,  and 
strange  nursery  stock,  many  previously  unheard-of  plant  diseases  have 
made  their  appearance.  As  the  farming  communities  have  become 
more  thickly  populated,  with  less  uncultivated  land  between  the  fields, 
these  diseases  have  spread  from  farm  to  farm  more  rapidly  than  in  the 
earlier  days,  and  the  losses  from  these  causes  have  been  so  heavy  during 
the  past  decade  that  the  farmers,  gardeners  and  orchardists  have  come 
to  the  Agricultural  Experiment  Stations  all  over  the  country  for  advice 
and  assistance  in  combating  their  troubles.  This  has  stimulated  an 
increased  interest  in  plant  diseases,  especially  along  bacteriological 
lines,  with  the  result  that  to-day  some  forty  bacterial  diseases  of  plants 
have  been  described. 

It  is  a  matter  of  not  infrequent  observation  that  closely  related 
species  of  plants,  as  well  as  animals,  exhibit  a  marked  difference  in  their 
susceptibility  to  the  same  disease-producing  agents.  The  Bartlett 
pear,  for  example,  suffers  more  severely  from  blight  than  the  Kieffer, 
and,  among  apples,  the  Toleman  Sweet  more  than  the  Rome;  the  small- 
leaf,  stemmy  varieties  of  tobacco  seem  to  be  more  resistant  to  the  Gran- 

*  Prepared  by  W.  G.  Sackett,  except  a  protozoal  disease  "Fingers  and  Toes"  by  J.  L.  Todd. 

949 


950  MICROBIAL   DISEASES    OF    PLANTS 

ville  wilt  than  the  large-leaf  kinds.  Resistance  of  this  sort,  which  ap- 
pears to  be  nothing  other  than  a  natural,  inborn  quality,  may  be  des- 
ignated as  natural  immunity,  and  it  is  immunity  of  this  kind  which 
plant  breeding  for  disease  resistance  has  secured.  A  good  illustration 
of  this  is  to  be  found  in  the  wilt-resistant  water  melon  of  the  Carolinas, 
which  is  the  result  of  crossing  a  naturally  susceptible  water  melon  with  a 
naturally  resistant  citron. 

Acquired  immunity  in  the  plant  world  is  a  field  yet  to  be  explored. 
Cases  have  been  cited  in  which  active  immunity  appears  to  have  followed 
the  disease,  but  these  are  extremely  rare  and  the  evidence  is  very 
questionable.  Passive  immunity,  at  the  present  time,  is  unknown. 


CHAPTER  I 

BLIGHTS 

STEM  BLIGHT  or  ALFALFA 
Pseudomonas  medicaginis — Sackett 

HISTORY  AND  DISTRIBUTION. — The  disease  has  been  known  in  Colo- 
rado since  1904  and  was  described  briefly  by  Paddock  in  1906  and  more 
fully  by  Sackett  in  1910.  It  is  distributed  generally  over  Colorado,  and 
is  reported  to  occur  in  Utah,  New  Mexico,  Arizona,  Nevada,  Nebraska 
and  Kansas. 

SYMPTOMS. — The  disease  is  primarily  a  stem  infection.  In  the 
earliest  stages,  the  stems  have  a  watery,  semi-transparent,  yellowish  to 
olive  green  appearance  along  one  side.  Soon  there  oozes  from  the  dis- 
eased tissue  a  thick,  clear,  viscid  liquid  which  spreads  over  the  surface 
and  collects  here  and  there  in  little  bead-like  droplets.  The  exudate  al- 
so dries  in  a  short  time  with  a  glistening  finish,  and  gives  the  stems  very 
much  the  appearance  of  having  been  varnished,  and  where  the  liquid 
has  collected  in  little  amber-colored  scales  and  has  hardened,  it  looks 
as  if  the  varnish  had  run  and  dried.  Stems  in  this  condition  have  a  dry, 
slightly  rough  feel  to  the  touch.  The  exudate  also  dries  uniformly  over 
the  surface  or  just  beneath  it,  and  there  produces  a  dark  brown,  resinous 
surface  which  blackens  with  age.  Such  stems  are  very  brittle  and 
easily  broken,  which  fact  makes  it  almost  impossible  to  handle  the 
crop  without  an  immense  amount  of  shattering  The  leaves  attached 
to  the  blighted  stems  usually  show  the  disease,  and  sometimes  they 
exhibit  the  infection  independent  of  the  stem.  In  this  case,  the 
petioles  become  watery  and  pale  yellow,  then  droop.  The  malady 
may  be  confined  to  the  petiole  and  base  of  the  leaflet,  or  it  may  involve 
the  whole  of  the  blade.  Occasionally  leaves  are  found  where  the 
inoculation  has  been  made,  apparently,  in  the  margin  of  the  leaflet, 
and  the  infection  has  proceeded  toward  the  middle.  In  such  instances, 
the  tender  tissue  has  a  watery  look,  as  if  it  had  been  bruised. 

951 


952  MICROBIAL  DISEASES    OF   PLANTS 

One-year-old  plants  may  exhibit  blackened  areas  in  the  crown,  and 
black  streaks  which  run  down  into  the  tap  root.  As  the  plant  grows 
older,  this  blackening  increases  until  the  whole  crown  becomes  in- 
volved, and  either  the  crown  buds  are  destroyed  or  the  root  is  no 
longer  able  to  perform  its  functions,  and  the  plant  dies. 

So  far  as  our  present  observations  go,  the  disease  appears  to  run  its 
course  with  the  first  cutting,  and  those  plants  which  have  sufficient 
vitality  throw  out  a  good  growth  for  the  second  and  third  cuttings. 

CAUSE  OF  THE  DISEASE. — If  a  small  piece  of  the  yellowish  green, 
watery  tissue  from  a  diseased  plant,  or  a  fragment  of  the  dried  exudate 
is  placed  in  a  drop  of  clean  water  on  a  glass  slide,  there  will  appear  on  all 


FIG.  195. — Pseudomonas  medicaginis.     Twenty-four  hour  culture  on  nutrient  agar; 
stained  with  aqueous  fuchsin;  Xiooo.     (Original.) 

sides  of  it,  after  half  a  minute,  a  dense,  milky  cloud,  which  can  be  seen 
readily  with  the  naked  eye,  and  which  slowly  diffuses  out  into  the  drop. 
When  this  preparation  is  examined  under  the  low  power  of  the  micro- 
scope, this  milky  zone  easily  resolves  itself  into  swarms  of  motile 
bacteria. 

The  organism  grows  readily  upon  the  ordinary  culture  media  and 
pure  cultures  of  the  germ,  inoculated  into  scarified  stems  of  healthy  al- 
falfa plants,  produce  the  disease  in  seven  to  nine  days  with  typical 
symptoms. 


BLIGHTS  953 

METHOD  ov  INFECTION. — Under  field  conditions  the  causal  organ- 
ism which,  presumably,  lives  in  the  soil,  enters  the  plants  early  in  the 
growing  season  with  soil  through  stems  which  are  cracked  and  split  by 
late  freezing.  In  some  instances,  inoculation  appears  to  take  place  by 
stomatal  and  water  pore  infections. 

CAUSAL  ORGANISM.— The  writer  has  given  the  name  Ps.  medicaginis  to  the 
causal  organism,  the  characteristics  of  which  are  as  follows:  It  is  a  short  rod  with 
rounded  ends,  about  i.2/t  to  2.4/1  by  0.5/4  to  o.8/i  the  majority  being  2.i/*  by  0.7/1. 
It  is  actively  motile  by  i  to  4  bi-polar  flagella;  non-spore  forming  and  non-capsule 
forming.  Filament  formation  occurs  frequently.  The  organism  stains  readily 
with  the  aqueous  stains,  but  is  Gram-negative. 

It  produces  a  surface  pellicle  on  broth.  Shining,  grayish  white  on  nutrient  agar, 
becomes  fluorescent  green  after  three  days.  Gelatin  stab,  surface  growth  only,  and 
no  liquefaction.  Potato  discolored,  moderate  growth,  cream  to  light  orange  yellow, 
starch  not  destroyed.  No  growth  in  Cohn's  solution.  Good  growth  in  Uschinsky's 
solution.  Plain  milk  shows  no  change.  Litmus  milk  becomes  bluer  after  seven 
days,  no  curd  and  no  peptonization  in  thirty  days.  No  indol.  No  hydrogen  sul- 
phide. Ammonia  produced  from  asparagin  solution,  Dunham's  solution  and 
nutrient  broth,  but  not  from  nitrate  broth.  Nitrates  not  reduced.  No  gas  and 
no  acid  from  dextrose,  etc.  Obligative  aerobe.  Optimum  temperature  28°;  no 
growth  at  37.5°.  Thermal  death-point  49.0°  to  50.0°.  Habitat,  soil.  Pathogenic 
for  alfalfa  (Medicago  saliva). 

CONTROL. — The  only  practical  way  of  combating  and  controlling  the 
blight  is  by  the  introduction  of  resistant  varieties,  but  no  entirely 
resistant  strain  has  been  obtained  up  to  the  present  time,  although  the 
Grimm  alfalfa  is  practically  free  from  it. 

As  a  means  of  control,  the  writer  recommends  that  the  frosted  al- 
falfa be  clipped,  as  soon  as  there  is  reasonable  certainty  that  danger 
from  late  frosts  is  past.  This  will  rid  the  plants  of  the  diseased  por- 
tions, and  afford  an  opportunity  for  the  early  growth  of  a  new  cutting. 
If  this  is  done  in  time,  the  regular  number  of  cuttings  should  be  secured 
with  little  or  no  loss  in  tonnage. 

BACTERIOSIS  OF  BEANS 

Pseudomonas  phaseoli — Erw.  Smith 

Frequently  the  foliage,  stems,  and  pods  of  the  common  beans,  as 
well  as  the  Lima  bean  are  attacked  by  a  bacterial  disease. 

SYMPTOMS. — The  pods  and  leaves  seem  to  furnish  the  best  food 
supply  for  the  microorganism,  and  it  is  here  that  we  find  the  most 


954  MICROBIAL  DISEASES    OF  PLANTS 

typical  lesions  developing.  Small,  reddish  spots  appear  which  in- 
crease rapidly  in  size  and  develop  into  watery,  amber-colored  blisters, 
surrounded  by  a  pink  or  reddish  border.  These  blisters  are  filled  with 
myriads  of  bacteria,  and  in  time,  they  dry  down,  forming  a  pale  yellow 
or  amber-colored  crust  over  the  affected  areas.  Ultimately  the  dis- 
eased leaves  become  brittle,  ragged,  and  are  worthless,  while  the  pods 
curl,  shrivel,  and  rot. 

METHOD  or  INFECTION. — It  is  believed  that  the  disease  is  intro- 
duced with  the  seed,  and  when  once  established,  is  spread  from  plant 
to  plant  by  rain,  dew,  and  leaf-eating  insects. 

CAUSAL  ORGANISM. — Ps.  phaseoli  Smith,*  is  a  short,  motile  rod  with  rounded 
ends,  which  produces  a  characteristic  yellow  growth  on  the  different  culture  media. 
Gelatin  slowly  liquefied.  Milk  becomes  slowly  alkaline,  casein  is  precipitated  by 
lab  ferment  and  partially  redissplved.  Very  marked  diastatic  action  on  potato 
starch.  No  gas  from  glucose,  saccharose,  etc.  Aerobic.  Uschinsky's  solution, 
growth  feeble  and  retarded.  Thermal  death-point  49.5°. 

CONTROL. — Care  should  be  taken  to  select  seed  from  healthy  fields 
where  the  disease  has  never  occurred.  The  disease  has  been  partially 
controlled  by  spraying  with  Bordeaux  mixture  when  the  plants  were 
2  to  3  inches  high,  again  ten  days  later,  and  after  blossoming. 

BLIGHT  OF  LETTUCE 

Ps.  mridilimdum — Brown 

The  disease  has  been  reported  recently  from  the  lettuce-growing 
sections  of  Louisiana,  and  is  described  as  producing  a  shriveled,  dried, 
burned  aspect  of  the  outer  leaves,  some  of  which  may  be  in  a  soft, 
rotted  condition.  The  deeper  leaves  exhibit  numerous  separate  or 
fused  spots  with  a  water-soaked  appearance;  the  center  of  the  head  is 
not  necessarily  involved. 

CAUSAL  ORGANISM. — Miss  Nellie  A.  Brownfhas  described  the  causal  organism, 
Ps.  viridilividum,  as  a  short  rod  with  rounded  ends,  motile  by  1-3  polar  flagella; 
stains  readily  with  the  ordinary  stains;  is  Gram-negative.  No  spores  have  been 
observed.  In  young  agar  cultures,  the  growth  is  cream-white  mottled  with  yellow, 
the  mottling  disappearing  with  age.  Gelatin  is  liquefied  slowly.  Nutrient  broth 

*  Smith,  Erw.,  Proc.  Am.  Asso.  Adv.  Sci.,  46,  228-290,  1897. 

f  Brown,  Nellie  A.,  "A  Bacterial  Disease  of  Lettuce,"  Jour.  Agr.  Res.,  Vol.  IV.,  No.  5. 
P-t47S,  1915. 


BLIGHTS  955 

is  clouded  and  becomes  lime-green  in  color  after  ten  days.  On  potato  it  produces 
a  characteristic  transient  dark-blue  green  color  which  develops  promptly  and  dis- 
appears on  the  sixth  day  or  earlier.  Growth  develops  readily  in  Uschinsky's  and 
Fermi's  solutions  changing  them  to  a  pale  green  color  in  three  to  five  days;  faint 
growth  occurs  in  Cohn's  solution.  Plain  milk  is  cleared  without  coagulation,  the 
cleared  fluid  becoming  a  pale  turtle-green  color;  litmus  milk  becomes  deeper  blue. 
Gas  is  not  produced  from  the  ordinary  sugars  in  Dunham's  solution.  Nitrates 
are  not  reduced,  and  some  indol  is  formed. 

METHOD  or  INFECTION. — Inoculation  experiments  indicate  that  in- 
fection may  take  place  either  through  the  stomata  or  through  wounds 
produced  by  mechanical  injury. 

CONTROL. — No  control  measures  have  been  reported. 


BLIGHT  OF  MULBERRY 
Pseudomonas  mori — Boyer  and  Lambert  (Smith) 

HISTORY. — The  disease  was  first  studied  in  1890  by  Cuboni  and 
Garbini  in  Italy,  and  later  by  Boyer  and  Lambert  in  France  who 
named  the  causal  organism  Bad.  mori,  but  did  not  describe  it.  In 
1908,  Erwin  F.  Smith*  found  a  similar  disease  in  some  of  the  Southern 
States,  and  described  the  causal  organism. 

SYMPTOMS. — According  to  Erwin  Smith,  the  blight  attacks  the 
leaves  and  young  shoots  of  the  mulberry,  producing  first  water-soaked 
spots,  which  later  become  sunken  and  black;  "foliage  more  or  less 
distorted;  shoots  soon  show  sunken  black  stripes  and  dead  terminal 
portions.  Action  of  disease  rather  prompt."  In  very  young  shoots, 
wood,  pith  and  bark  are  invaded  by  bacteria;  in  older  shoots  the  germs 
are  confined  mostly  to  the  xylem. 

CAUSAL  ORGANISM. — The  organism  is  a  rod  with  rounded  ends,  3.6/1  by  1.2/1, 
motile  by  i  to  2  polar  flagella,  attached  to  one  end.  No  spores  observed;  pseudo- 
zoogloea  occur.  Stains  readily  with  carbol  fuchsin;  Gram-negative. 

On  agar,  spreading,  smooth,  dull,  translucent,  shiny,  white;  medium  not  stained. 

On  potato,  spreading,  glistening,  smooth,  white  to  dirty  white,  shiny,  medium 
grayed,  slight  action  on  starch.  Gelatin  stab,  filiform,  no  liquefaction.  Beef  broth, 
pellicle,  strong  clouding.  Milk,  no  coagulation,  rendered  alkaline,  becomes  clear  by 
solution  of  fat  and  casein,  litmus  not  reduced.  No  growth  or  scant  in  Cohn's 
solution.  Uschinsky's  solution,  copious,  pellicle,  not  viscid  fluid,  bluish-fluorescent 
color.  No  gas  from  dextrose,  saccharose,  etc.  Aerobic.  No  indol  or  slight. 
Nitrates  not  reduced.  Thermal  death-point  51.5°;  does  not  grow  at  37°. 

*  Smith,  Erwin  P.:  Bacterial  Blight  of  Mulberry,  Science  N   S..  Vol.  XXXI,  803. 


956  MICROBIAL  DISEASES    OF   PLANTS 

BLADE  BLIGHT  or  OATS 
Pseudomonas  avena — Manns  and  Bacillus  avetuz — Manns* 

HISTORY  AND  DISTRIBUTION. — A  specific  bacterial  disease  of  oats 
has  been  described  by  Manns  in  1909.  What  appears  to  have  been  a 
similar  trouble,  extending  from  the  Atlantic  coast  west  to  Indiana,  and 
from  the  Great  Lakes  to  the  Gulf  States,  was  observed  as  early  as  1890 
by  Galloway  and  South  worth.  Its  appearance  was  noted  for  the  first 
time  in  Colorado  in  1915. 

SYMPTOMS. — In  the  early  stages  of  the  disease  there  is  "a  yellowing, 
beginning  either  as  small  round  lesions  on  the  blade,  or  as  long,  streak 
lesions  extending  throughout  the  blade  or  even  the  whole  length  of  the 
culm  and  blades.  In  the  advanced  stages,  the  affected  blades  take  on  a 
mottled  to  almost  red  color,  which  has  been  called  'rust'  and  'blight.' " 

CAUSE  OF  THE  DISEASE. — The  disease  is  produced  by  the  symbiotic 
growth  of  two  bacteria  whose  activity  is  favored  by  rainy,  humid,  and 
cloudy  weather.  One  of  these  organisms,  Ps.  avence,  alone,  is  said  to  be 
capable  of  effecting  the  blight  in  a  mild  form,  while  the  other,  B. 
avena,  is  nonpathogenic;  but  a  mixture  of  the  two  germs  results  in  an 
aggravated  attack. 

METHOD  OF  INFECTION. — Infection  takes  place  through  the  stomata, 
the  organisms  being  spattered  on  the  leaves  from  the  soil  by  rains. 
Grain  insects  are  also  responsible  for  spreading  the  disease. 

CONTROL. — It  is  believed  that  the  control  of  the  disease  lies  in  the 
selection  of  resistant  strains. 

STEM  BLIGHT  OF  FIELD  AND  GARDEN  PEAS 
Pseudomonas  pisi — Sackett 

HISTORY  AND  DISTRIBUTION. — The  disease  occurs  in  several  of  the 
Western  States,  particularly  in  the  mountain  valleys  of  the  higher 
altitudes.  It  was  first  observed  in  Colorado  in  1915,  where  it  caused  a 
loss  of  approximately  one-third  of  the  field  peas  in  the  San  Luis  Valley, 
while  in  other  parts  of  the  State  where  garden  peas  are  grown  for  can- 
ning purposes,  the  crop  was  materially  affected. 

SYMPTOMS. — The  plants  usually  show  the  infection  before  they  are 
8  inches  high,  and  many  succumb  before  they  reach  that  size.  Both 

*  Manns,  "The  Blade  Blight  of  Oats,  A  Bacterial  Disease,"  Bull.  210,  Ohio  Exp.  Sta.,  1909. 


BLIGHTS  957 

field  and  garden  peas  are  affected  alike,  and  the  symptoms  simulate 
the  bacterial  stem  blight  of  alfalfa.  The  stems  have  a  watery,  olive- 
green  appearance  which  soon  becomes  olive-brown,  and  in  the  last 
stages  dark  brown.  The  leaves  and  stipules  appear  watery  at  first,  as 
if  bruised,  and  later  turn  ocher  yellow  in  color;  this  is  often  accompanied 
by  wilting.  In  young  plants,  the  discoloration  of  the  stems  is  followed 
by  a  shrivelling,  and  ultimately  the  plants  dry  up  and  die;  in  the  older 
ones,  where  the  infection  has  taken  place  later,  the  same  condition  may 
result,  but  on  the  whole,  the  disease  appears  to  be  less  serious,  and  in 
some  cases  the  plants  seem  to  outgrow  the  blight.  Frequently  when 
the  first  and  earliest  shoots  are  destroyed,  the  plant  throws  up  new 
shoots  from  below  ground,  and  a  good  late  crop  is  obtained,  in  spite 
of  the  trouble. 

CAUSAL  ORGANISMS. — Pseudomonas  pisi,  n.  sp.,  as  described  by  Sackett,* 
is  a  short  rod  with  rounded  ends,  motile  by  means  of  a  single  polar  flagellum; 
neither  spores  nor  capsules  observed;  filaments  formed  commonly;  stains  readily 
with  aqueous  stains,  and  is  Gram-negative. 

It  produces  a  flaky  surface  scum  with  heavy  clouding  in  broth.  On  nutrient 
agar  the  growth  is  smooth,  glistening,  grayish  white,  and  the  medium  is  not  dis- 
colored. Gelatin  is  liquefied  rather  rapidly.  On  potato,  smooth,  glistening,  cream 
to  orange-yellow;  medium  becomes  grayish  brown.  No  growth  in  Cohn's  or 
Uschinsky's  solutions.  Heavy  clouding  with  white  surface  pellicle  in  Fermi's 
solution;  clouding  with  surface  scum  in  FraenkePs  solution;  slight,  transient  cloud- 
ing in  Naegli's  solution.  Plain  milk  is  coagulated,  and  the  coagulum  is  slowly 
peptonized,  the  supernatant  liquid  becoming  yellowish  green.  Litmus  milk  becomes 
bluer,  and  the  litmus  is  reduced,  the  liquid  becoming  greenish-gray.  Neither 
indol  nor  hydrogen  sulphide  is  produced.  Ammonia  is  produced  from  asparagin 
and  peptone.  Nitrates  are  not  reduced.  No  gas  is  formed  from  sugars,  but  acid  is 
produced  from  dextrose,  saccharose  and  galactose.  Obligative  aerobe.  Optimum 
temperature  25°  to  28°.  Thermal  death-point  50°.  Habitat,  soil. 

Pathogenic  for  field  pea  and  garden  pea  (Pisum  sativum  var.  arvense  and  Pisum 
sativum}. 

METHOD  OF  INFECTION. — Experimental  inoculations  indicate  that 
infections  take  place  either  through  the  stomata  or  through  wounds 
produced  by  mechanical  injuries. 

CONTROL. — There  seems  to  be  a  close  relation  between  the  preva- 
lence of  the  disease  and  a  late,  cold  spring.  The  low  temperatures 
appear  to  make  the  plants  more  susceptible,  and  as  a  result  the  early 

*  Sackett,  Walter  G.,  "Stem  Blight  of  Field  and  Garden  Peas — A  Bacterial  Disease," 
Bull.  218,  Colorado  Exp.  Sta.,  April,  1916. 


958 


MICROBIAL  DISEASES    OF   PLANTS 


plantings  are  the  worst  affected.     As  a  control  measure,  planting  from 
two  to  three  weeks  later  is  suggested. 

PEAR  BLIGHT 

Bacillus    amylovorus — (Burrill)    De    Toni 

HISTORY  AND  DISTRIBUTION. — As  early  as  1780,  William  Denning, 
a  fruit  grower,  who  lived  on  the  Highlands  of  the  Hudson  River,  ob- 
served pear  blight  in  the  trees  of  his  neighborhood.  It  is  very  probable 


FIG.  196.— Two  pear  twigs.     The  upper  one  affected  with  Fire  Blight,  the  lower  one 
healthy.     (After  Sackett,  Mich.  Agr.  Exp.  Sla.) 

that  blight  existed  many  years  before  this  in  eastern  North  America  on 
some  of  our  native  wild  crabs,  hawthorns,  and  wild  plums,  and  with  the 
introduction  of  cultivated  varieties,  it  found  a  new  field  for  attack.  As 
the  farming  communities  became  more  thickly  populated,  and  the 
orchards  more  numerous,  it  has  spread  gradually  westward  over  the 


BLIGHTS  959 

Allegheny  Mountains  into  the  Mississippi  Valley,  across  the  Great 
Plains,  and  over  the  Rocky  Mountains  to  the  Pacific  Coast.  So  gen- 
erally is  it  distributed  over  the  United  States  and  Canada  that  a  blight- 
free  orchard  is,  indeed,  a  rare  sight.  The  disease  has  progressed  with 
such  severity  that,  to-day,  commercial  pear  growing  in  Colorado  has 
been  practically  abandoned,  and  the  industry  in  California  is  being 
threatened  with  destruction.  So  far  as  our  present  knowledge  goes, 
the  blight  is  of  American  origin  and  is  confined  to  North  America. 

OCCURRENCE. — While  the  ravages  of  the  disease  are  worst  upon  the 
pear,  from  which  fact  the  disease  derives  its  name,  many  varieties  of  the 
apple,  quince,  apricot  and  plum,  together  with  the  mountain  ash,  service 
berry,  wild  crabs  and  several  species  of  hawthorn,  have  suffered  severely 
from  the  same  cause,  and  are  capable  of  transmitting  the  disease  from 
one  to  the  other. 

SYMPTOMS. — The  disease  is  most  easily  recognized  during  the  grow- 
ing season,  when  it  attacks  the  blossom  clusters  and  the  tips  of  the 
growing  twigs.  In  this  form  it  is  known  as  blossom  and  twig  blight.  The 
leaves  attached  to  these  parts  usually  turn  brown  or  black,  either  wholly 
or  in  part,  the  petioles  blacken,  and  the  young  twigs  show  a  blackened, 
shriveled  bark,  having  much  the  appearance  of  green  brush  which  has 
been  burned  only  partially.  It  is  from  these  symptoms  that  we  get 
the  name  Fire  Blight,  so  appropriately  applied  to  pear  blight.  The 
blackened,  withered  leaves  cling  tenaciously  to  their  blighted  twigs 
long  after  the  other  leaves  have  fallen  in  the  fall,  and  in  this  way  afford 
the  orchardist  an  easy  way  of  recognizing  the  blighted  areas. 

Frequently  the  disease  finds  its  way  into  the  larger  limbs  and  even 
the  trunk  of  the  tree,  where  it  produces  body  blight.  This  form  is 
characterized  in  the  early  stages  by  a  cracking  of  the  bark  and  the 
oozing  of  a  thick,  dirty  white  or  brown,  sticky  liquid  which  collects  here 
and  there  in  drops  over  the  injured  surface.  As  the  disease  progresses, 
the  splitting  of  the  bark  increases  and  the  area  involved  becomes  rough, 
giving  rise  to  a  canker.  This  is  not  to  be  confused  with  sun  scald,  in 
which  the  bark  dries  down  and  adheres  firmly  to  the  wood  beneath,  and 
which  is  due  to  an  entirely  different  cause. 

The  immature  fruit  manifests  the  blight  by  turning  black,  shriveling 
and  taking  on  a  dried,  mummified  appearance.  Accompanying  these 
changes,  drops  of  a  thick,  sticky  exudate  usually  appear  on  the  surface. 

If  a  cross  section  is  made  of  a  diseased  twig  or  limb,  one  invariably 


960  MICROBIAL   DISEASES    OF   PLANTS 

finds  a  blackened  ring  in  the  region  of  the  cambium  layer.  This 
phenomenon,  the  significance  of  which  will  be  explained  later,  serves 
as  a  reasonably  reliable  means  of  diagnosis. 

CAUSE. — A  microscopic  examination  of  either  the  blackened  cam- 
bium or  a  drop  of  the  exudate  shows  swarms  of  motile  rods,  B.  amylo- 
vorus,  which  Burrill  of  the  University  of  Illinois,  as  early  as  1878, 
credited  with  being  the  cause  of  pear  blight.  By  inoculating  healthy 
trees  with  this  gummy  material,  he  was  able  later  to  demonstrate  his 
point  experimentally,  and  with  his  work  and  that  of  a  Dutch  botanist, 
Wakker,  we  have  the  beginning  of  the  study  of  bacterial  diseases  of 
plants. 

METHODS  OF  INFECTION. — The  more  careful  observers  believe  that 
insects,  especially  bees,  plant  lice  and  twig  borers  are  responsible  for  the 
initial  infection  and  subsequent  spread  of  the  disease.  It  has  been 
found  that  the  bacteria  find  protection  from  the  adverse  conditions  of 
winter  in  the  margins  of  the  old  cankers  next  to  the  sound  bark,  and 
also  in  some  of  the  blighted  shoots  and  twigs.*  These  hold-over 
bacteria  become  active  with  the  increased  flow  of  sap  and  the  higher 
temperature  of  spring,  and  soon  spread  into  the  adjacent  healthy 
bark.  Here  they  multiply  so  rapidly  that  at  about  the  timef  the  trees 
are  in  blossom,  they  begin  to  ooze  from  the  cracks  in  the  diseased  bark 
as  drops  of  a  thick,  sticky  material,  dirty  white  or  brown  in  color. 
Insects  are  attracted  to  this  ooze,  apparently  feed  upon  it,  smear  their 
feet,  bodies  and  mouth  parts,  and  then  fly  away  to  the  opening 
blossoms.  Here  they  feed  upon  the  nectar  and  while  so  doing  infect  the 
flowers.  The  germs  increase  rapidly  in  this  sweet  liquid,  and  each  bee 
that  visits  the  flower  subsequently  carries  away  millions  of  germs  to 
infect  other  blossoms.  From  the  flowers,  the  bacteria  find  their  way 
into  the  cambium  and  softer  tissues  of  the  bark,  where  the  disease  is 
confined  almost  entirely.  After  about  ten  days  the  progress  of  the 
germs  can  be  noted  by  the  blackening  of  the  flower  clusters,  and  the 
wilting  and  blackening  of  the  leaves  of  the  fruit  spurs.  Following  the 
collapse  of  the  fruit  spurs,  the  disease  may  move  down  the  twig  an 
inch  or  more  a  day,  causing  it  to  appear  watery,  turn  black  and  shrivel. 
The  blackening  may  be  10  to  12  inches  behind  the  advancing  infection. 

*  The  writer  examined  a  number  of  blighted  pear  twigs  Apr.  14,  19x1.  collected  from  different 
orchards  in  Colorado  and  found  B.  amylovorus  alive  in  23.53  per  cent.  The  germs  occurred  in 
the  2  cm.  adjacent  to  the  healthy  part  of  the  twigs. 

t  Whetzel.  Bull.  272.  Cornell  Exp.  Station,  1909. 


BLIGHTS  961 

This  may  continue  until  the  whole  limb  becomes  involved,  but  as  a  rule 
it  is  only  the  smaller  twigs  which  are  the  worst  affected.  From  this 
it  will  be  seen  that  the  external  blackening  cannot  be  relied  upon,  early 
in  the  season  at  least,  as  a  guide  to  the  exact  location  of  the  disease; 
however,  as  the  season  advances,  the  plant  tissues  harden,  conditions 
for  germ  life  become  less  favorable,  and  as  a  result,  by  the  middle  of 
summer,  the  active  progress  of  the  blight  is  checked  by  natural  causes, 
and  the  blackening  overtakes  the  advancing  infection. 

Blight  which  appears  on  the  water  sprouts  of  large  limbs  later  can 
usually  be  accounted  for  by  inoculation  by  plant  lice  and  the  pear  twig 
borer. 

CAUSAL  ORGANISM. — According  to  Jones*  Bacillus  amylovorus  possesses  the 
following  characteristics:  Short  motile  bacillus,  rounded  ends,  i/i-i.8/i  by  0.5/1- 
o.g/z;  stains  readily  with  the  aqueous  stains;  Gram-negative.  No  spores  observed. 

Agar  slant  and  potato,  growth  moderate,  filiform,  glistening,  smooth,  grayish 
white,  semi-opaque,  butyrous.  Gelatin  stab,  growth  rather  slow,  filiform,  slight 
crateriform  liquefaction  after  twenty  days.  Nutrient  broth,  moderate  clouding, 
uniform;  if  left  undisturbed,  a  delicate  pellicle  or  ring  may  form  which  breaks  up  and 
sinks  with  the  slightest  jar;  scant  finely  granular  sediment  after  ten  days.  Litmus 
milk,  light  blue  in  four  days,  pinkish  in  six  days,  light  blue  again  in  twelve  days, 
upper  layer  blue  in  eighteen  days;  soft  gelatinous  curd  six  to  ten  days,  with  whey 
on  the  surface.  Cohn's  solution,  no  growth.  Uschinsky's  solution,  no  growth. 
Nitrates  not  reduced.  No  indol.  Thermal  death-point  50°.  Optimum  tem- 
perature 23°  to  25°.  Slight  acid  production  but  no  gas  from  dextrose,  etc.  Starch 
is  not  fermented. 

CONTROL. — It  is  obvious  that  spraying  is  useless  for  a  disease  of  this 
character,  where  the  germs  are  located  beneath  the  surface. 

A  systematic  cutting  out  of  the  diseased  limbs  and  twigs  wherever 
and  whenever  they  appear  is  the  only  practical  method  of  controlling 
the  blight.  It  is  almost  impossible  to  get  all  of  the  diseased  material 
in  the  summer  time  when  the  heavy  foliage  hides  it,  but  in  the  fall  and 
winter  the  blighted  branches  can  be  recognized  very  readily  by  the  tufts 
of  dead  leaves  clinging  to  them.  It  is  necessary  in  removing  the  dead 
wood  to  cut  well  below  the  discolored  part,  10  to  15  inches,  for 
the  bacteria  may  be  considerably  in  advance  of  the  discolored  area. 
Clean  out  all  old  cankers  by  cutting  well  into  the  healthy  part  and  by 
removing  the  dried,  diseased  material.  Disinfect  the  freshly  cut  sur- 
faces of  this  wound  as  well  as  the  exposed  ends  of  twigs  and  limbs  with 

*  Jones,  D.  H..  The  Bacterial  Blight  of  Apple,  Pear  and  Quince  Trees.     Bull.  176,  Ontario 

Agr.  College. 
61 


962  MICROBIAL   DISEASES    OF   PLANTS 

i :  1000  solution  of  mercuric  chlorid.     All  diseased  wood  must  be  collected 
and  burned. 

STREAK  DISEASE  or  SWEET  PEAS  AND  CLOVERS 
Bacillus  lathyri — Manns  and  Taubenhaus 

HISTORY. — The  first  recorded  observations  of  this  disease  were  made 
by  Diggs  on  sweet  peas  in  Dublin,  Ireland,  in  1904.  The  trouble  was 
known  locally  as  "Streak"  disease  of  the  sweet  pea,  and  various 
parasitic  fungi  were  assigned  as  the  cause.  One  investigator  even 
ventured  the  assertion  that  the  malady  was  of  a  physiological  nature. 
In  1912,  Taubenhaus  isolated  a  bacillus  from  clovers  and  sweet  peas 
collected  in  the  vicinity  of  Newark,  Delaware,  and  which  bore  lesions 
similar  to  those  described  for  "Streak."  Subsequent  inoculations 
with  pure  cultures  proved  the  disease  to  be  of  bacterial  origin  and 
identical  with  that  observed  in  England  and  Ireland. 

SYMPTOMS. — The  disease  makes  its  appearance  during  the  season  of 
heavy  dew  and  is  characterized  by  light  reddish-brown  to  dark  brown 
spots  and  streaks,  almost  purple  when  old,  along  the  stems.  They 
usually  originate  near  the  ground,  which  seems  to  indicate  distri- 
bution by  spattering  rain  and  infection  through  the  stomata.  The 
disease  is  quickly  distributed  over  the  more  mature  stems,  and  ulti- 
mately the  cambium  and  deeper  structures  are  destroyed  in  con- 
tinuous areas  resulting  in  the  premature  death  of  the  plant.  Occa- 
sionally the  petioles  and  leaves  show  the  infection;  the  latter  exhibit 
the  water-soaked. areas  common  to  bacterial  stomatal  infections  such 
as  are  met  with  in  alfalfa  blight  and  bacteriosis  of  beans. 

CAUSAL  ORGANISM. — Manns  and  Taubenhaus  have  described  the  organism 
which  is  responsible  for  the  disease  as  a  new  species  under  the  name  Bacillus  lathyri. 
It  is  a  small  rod,  motile  by  means  of  8-12  short,  peritrichiate  flagella;  it  grows  lux- 
uriantly upon  all  of  the  common  nutrient  media,  especially  if  sugars  are  present, 
producing  a  yellow  pigment;  on  glucose  agar,  colonies  appear  in  twenty-four  to 
thirty-six  hours,  showing  a  tendency  to  become  stellate  or  auriculate. 

PATHOGENESIS. — Bacillus  lathyri,  n.  sp.  has  been  isolated  from 
specific  lesions  on  the  following  hosts:  Sweet  pea,  Lathyrus  spp., 
red,  alsike  and  mammoth  clovers,  soy  beans,  garden  beans,  cow  peas 
and  alfalfa. 

METHOD  OF  INFECTION. — Infection  appears  to  take  place  through 
the  stomata,  the  organism  being  spattered  on  the  plants  from  the 
soil_duringrrains. 


BLIGHTS  963 

CONTROL. — On  small  areas,  heavy  mulching  of  straw  along  either 
side  of  the  row  is  suggested  as  a  possible  means  of  preventing  the 
distribution  of  the  disease. 

TOMATO  BLIGHT 
Bacterium  (?)  michiganense — Erw.  Smith* 

The  disease  is  distinct  from  the  wilt,  caused  by  B.  solanacearum,  in 
that  there  is  not  the  sudden  collapse  of  the  whole  plant,  but  rather  a 
slow  yellowing  or  wilting  of  the  leaves,. one  at  a  time.  The  causal 
organism  produces  cavities  in  the  pith  and  bark  as  well  as  in  the 
vascular  system. 

WALNUT  BLIGHT  OR  BACTERIOSIS 
Pseudomonas  juglandis — Pierce 

HISTORY  AND  DISTRIBUTION. — Attention  was  first  called  to  this  dis- 
ease as  it  occurred  in  California  by  Pierce  f  in  1893  although  it  had 
been  observed  in  Los  Angeles  County  in  about  1891.  Outside  of 
California,  it  is  known  to  occur  in  Oregon,  Texas  and  midway  down 
the  Pacific  coast  of  Mexico.  What  appears  to  be  a  similar  trouble 
has  been  reported  from  New  Zealand  and  France. 

SYMPTOMS.  { — All  of  the  new,  tender,  growing  parts  of  the  tree, 
such  as  young  nuts  and  branches,  petioles  of  leaves,  midveins,  fine 
lateral  veins  and  adjoining  parenchyma  are  subject  to  the  attacks. 
On  the  branches,  the  disease  always  starts  in  the  young  succulent 
growth  and  manifests  its  presence  by  small,  discolored  areas  which 
under  favorable  conditions  may  extend  2  to  3  inches  along  the  green 
shoot.  As  the  infection  progresses,  the  central  portion  of  the  lesion 
turns  black  and  is  surrounded  by  a  water-soaked  margin.  In  the 
later  stages,  the  whole  diseased  area  becomes  blackened  and  in-  many 
instances  has  a  somewhat  shrunken,  dried-out,  deformed,  cracked 
appearance  due  to  the  drying  out  of  the  tissue.  In  severe  cases  the 
tissue  is  killed  inwardly  to  the  pith,  while  in  the  milder  attacks  only 
the  bark  and  wood  are  diseased.  As  the  wood  hardens,  the  infection 
is  checked,  and  the  vitality  of  the  tree  is  not  affected  to  any  extent,  the 

•  Smith,  Erw.,  Science,  N.  S.,  Vol.  XXXI,  803,  p.  794,  1910. 

t  Pierce,  N.  B.,  Bot.  Gaz.,  31;  272-273,  1901. 

t  Smith,  C.  O.t  "Walnut  Blight,"  Bull.  231,  Calif.  Exp.  Sta.,  320,  1912. 


964 


MICROBIAL  DISEASES    OF   PLANTS 


crop  suffering  rather  than  the  tree.  The  leaves  sometimes  exhibit  a 
blackening  or  browning  of  the  petioles  and  veins,  while  the  intermediate 
tissue  may  develop  brownish,  circular  or  angular  spots.  The  disease 
does  not  cause  serious  defoliation  of  the  tree.  The  catkins  are  probably 
not  affected.  It  is  upon  the  young  nuts  that  bacteriosis  is  especially 


FIG.  197. — Walnuts  affected  by  Bacteriosis,  mostly  stigma  or  blossom-end  infection. 
(After  C.  0.  Smith,  Calif,  Bull.  231.) 

destructive,  and  it  would  be  of  little  economic  importance  did  it  not 
attack  these.  Many  of  the  nuts  may  become  infected  and  fall  when 
they  are  K  to  J^  inch  in  diameter  and  continue  to  drop  throughout  the 
summer.  A  conservative  estimate  of  the  loss  places  it  at  50  per  cent 
in  badly  diseased  groves.  The  most  common  point  of  infection  is  at 
the  blossom  end,  although,  it  may  start  at  any  place  on  the  nut.  In 


BLIGHTS  965 

the  early  stage,  the  lesions  appear  as  small,  circular,  raised,  discolored, 
water-soaked  areas;  later,  these  spots  increase  in  size  and  turn  black. 
Under  favorable  conditions,  the  disease  may  extend  through  the  hull 
and  shell-forming  tissues  into  the  kernel  which  at  length  becomes 
blackened  and  finally  destroyed. 

CAUSAL  ORGANISM. — According  to  Smith,  C.  O.,  Pseudomonas  juglandis,  Pierce, 
is  a  rod  with  rounded  ends;  single  or  in  pairs,  rarely  in  chains;  measures  i-5/z  to  3.01/1*  X 
0.3/1  to  0.51/1;  stains  readily  with  the  ordinary  aniline  dyes;  Gram-positive;  spores  and 
capsules  not  observed;  motile  by  means  of  a  single  polar  flagellum;  agar  colonies 
nucleated,  circular,  moist,  shining,  pale  yellow  with  regular  margins;  startiform 
liquefaction  in  gelatin;  potato,  abundant,  moist,  shining,  slimy,  raised,  white  chang- 
ing to  yellow;  uniform  turbidity  and  ring  in  bouillon,  slight  flocculent  precipitate; 
indol  produced;  nitrates  not  reduced;  enzymes,  diastasic,  cytohydrolytic,  rennet, 
proteolytic;  milk  coagulated,  curd  digested;  litmus  milk  wine  colored;  viability, 
nine  and  one-half  months  on  potato;  methylene-blue  milk  reduced. 

METHOD  OF  INFECTION. — It  has  been  shown  that  the  causal 
organisms  live  over  winter  in  the  old  lesions  of  the  wood  and  bark  and 
that  in  the  spring  they  exude  to  the  surface  and  are  carried  to  the  new 
growth,  to  which  they  gain  entrance  through  the  stomata.  The  disease 
is  most  severe  during  seasons  when  the  fogs  and  rainfall  are  heaviest, 
and  in  those  localities  where  rain  and  fogs  are  abundant.  "  During 
one  of  these  fogs  the  trees  become  saturated,  water  dripping  from  one 
portion  of  the  tree  to  another  which  could  easily  carry  the  disease 
organisms  to  healthy  tissue."  Distribution  by  this  means  is  thought 
to  be  one  of  the  most  important,  if  not  the  most  important,  methods  of 
spreading  the  trouble.  Insects  probably  play  some  part  in  the  dis- 
semination of  blight. 

PATHOGENESIS. — Pathogenic  for  Juglans  regia  (English)  under 
natural  conditions;  pure  culture  inoculations  give  positive  lesions  on 
Juglans  nigra  (eastern  black),  Juglans  hindsii  (northern  Cal.  black), 
Juglans  calif ornica  (southern  Cal.  black),  Juglans  cinerea  (butternut). 

CONTROL. — Systematic  spraying  experiments  with  Bordeaux  mix- 
ture, lime-sulphur,  and  a  sulphur  spray  have  demonstrated  that  spray- 
ing is  impracticable  and  has  little  value  as  a  means  of  control.  Applica- 
tions of  lime  to  the  soil  have  resulted  in  no  benefit.  It  has  been 
observed  that  individual  trees  exhibit  great  differences  in  their  natural 
resistance  to  the  blight,  and  at  the  present  time  the  selection  and 
propagation  of  varieties  which  are  more  or  less  immune  promises 
the  most  practical  solution  to  the  problem. 


CHAPTER  II 
GALLS  AND  TUMORS 

CROWN  GALL 
Pseudomonas  tumefaciens. — Erw.  Smith  and  Townsend 

Crown  gall  is  one  of  the  most  recent  plant  diseases  to  be  traced  to 
bacterial  origin.  Its  occurrence  is  so  common  in  nursery  stock  that  in 
a  certain  Western  State,  75  per  cent  of  the  young  trees  and  shrubs 
condemned  by  nursery  inspectors  are  condemned  for  crown-gall,  and 
Tourney  places  the  annual  loss  to  orchardists  at  $500,000  to  $1,000,000. 


FIG.  198. — Crown  gall  with  hairy  root  on  nursery  stock.     Northern  Spy  apple. 

(After  Paddock.) 

HISTORY. — Smith  and  Townsend*  working  with  the  gall  of  the  Paris 
daisy  observed  bacteria  in  these  outgrowths  in  1904,  but  it  was  not 
until  1906  that  they  succeeded  in  isolating  the  causal  organism  and 

*  Smith,  Erw.  P.,  and  Townsend,  C.  O.,  "A  Plant  Tumor  of  Bacterial  Origin,"  Science,  N. 
S.  Vol.  XXV,  No.  643,  p.  671-673,  1907;  "The  Etiology  of  Plant  Tumors,"  Science,  N.  S. 
Vol.  XXX,  No.  763,  p.  233,  1909. 

Townsend,  C.  O.,  "A  Bacterial  Gall  of  the  Daisy  and  Its  Relation  to  Gall  Formations  on 
Other  Plants,"  Science,  N.  S.  Vol.  XXIX,  p.  273  (Abstract),  1909. 

966 


GALLS   AND    TUMORS  967 

in  securing  satisfactory  re-inoculations.  Subsequent  studies*  have 
shown  that  this  same  microorganism  is  responsible  for  the  pathological 
condition  that  we  recognize  as  crown  gall  in  its  various  forms  on  the 
different  hosts.  One  of  the  remarkable  things  about  this  disease  is 
the  large  number  of  families  which  are  subject  to  the  infection. 

PATHOGENESIS. — A  partial  list  of  the  plants  upon  which  crown  gall 
occurs  naturally  or  upon  which  it  has  been  produced  by  laboratory 
inoculation  includes  the  daisy,  tomato,  tobacco,  potato,  carnation, 
peach,  rose,  cabbage,  grape,  hop,  sugar-beet,  turnip,  red  beet,  carrot, 
radish,  chrysanthemum,  oleander,  marigold,  pyrethrum,  almond, 
clover,  white  poplar,  Persian  walnut,  Pterocarya,  gray  poplar,  cotton, 
alfalfa,  raspberry,  geranium,  apple,  willow,  quince. 

SYMPTOMS. — The  swellings  or  galls,  small  at  first,  usually  appear 
just  below  the  ground  line  (crown),  at  or  near  the  juncture  of  the  stock 
and  scion.  These  may  be  either  hard  or  soft  galls;  the  former  are 
smooth,  soft,  spongy,  white  to  flesh-colored  outgrowths  which  may 
reach  a  very  appreciable  size  during  one  season  and  then  be  entirely 
decomposed  and  disappear  by  the  following  spring;  the  latter  increase 
in  size  more  slowly,  persist  year  after  year,  harden  and  become  rough 
and  warty  on  the  surface  with  age.  Both  are  crown  galls  and  both  are 
produced  by  bacteria.  According  to  Smith,f  "  Whether  a  crown  gall 
shall  develop  as  a  hard  gall  or  a  soft  gall  would  seem  to  depend  chiefly 
if  not  altogether  on  which  meristem  cells  receive  the  initial  impulse. 
If  the  cells  first  infected  are  principally  the  mother  cells  of  medullary 
rays,  we  may  assume  that  the  gall  will  be  a  'soft  gall/  and  readily 
inclined  to  decay.  If,  on  the  contrary,  the  needle  or  other  carrier  of 
infection  wounds  principally  those  meristem  cells  which  give  rise  to 
tracheids  and  wood  fibers,  the  gall  will  be  a  'hard  gall,'  of  slow  growth 
and  long  duration."  The  structure  of  the  galls  is  unlike  that  of  club- 
root  of  cabbage  in  that  the  latter  is  an  hypertrophy  while  the  former 
is  an  hyperplasia.  Frequently  this  disease  assumes  a  form  known  as 
"hairy  root"  characterized  by  the  presence  of  bunches  or  tufts  of  closely 
matted  rootlets  with  enlargements  at  their  bases.  As  the  gallsj  enlarge, 
the  function  of  the  adjacent  conducting  tissue  is  interfered  with,  and 

*  Smith,  Erw.  P.,  Brown,  Nellie  A.,  Townsend,  C.  O.,  "Cr,own-gall  of  Plants:  Its  Cause 
and  Remedy,"  Bull.  213,  Bur.  Plant  Ind.,  U.  S.  Dept.  Agr..  1911. 

f  Smith,  C.  O.,  "Further  Proof  of  the  Cause  and  Infectiousness  of  Crown  Gall,"  Bull.  235, 
Calif.  Exp.  Sta.,  1912. 

t  Very  hairy  roots  often  accompany  these. 


968  MICROBIAL  DISEASES    OF   PLANTS 

the  circulation  is  impaired,  as  is  shown  by  the  poor  growth  and  dwarfed 
appearance  of  the  trees. 

The  development  of  this  disease  is  looked  upon  by  Smith*  and  his 
associates  as  paralleling  closely  what  takes  place  in  cancer  in  man  and 
animals.  The  primary  tumors  have  been  observed  to  send  out  "roots" 
or  tumor-strands  for  some  distance  into  the  normal  tissue,  and  from 
these  tumor-strands,  secondary  tumors  may  arise  which  tend  to  take 
on  the  structure  of  the  primary  tumor,  e.g.,  "if  the  latter  is  in  the  stem 
and  the  former  in  a  leaf,  the  secondary  tumor  shows  a  stem  structure." 
"There  are  no  metastases  in  crown  gall  *  *  *  for  whether  a  cancer 
shall  be  propagated  by  floating  islands  of  tissue,  or  only  by  tumor- 
strands,  appears  to  be  a  secondary  matter  depending  upon  the  char- 
acter of  the  host  tissues  rather  than  the  nature  of  the  disease."  The 
salient  point  is  the  internal  stimulus  to  cell  division  which  arises  from 
the  presence  of  the  microorganisms  within  certain  cells. 

METHOD  OF  INFECTION. — Little  is  known  about  the  natural  channels 
of  infection,  but  inoculation  through  wounds  induced  by  poor  grafting, 
careless  cultivation,  and  by  borers,  nematodes,  etc.,  is  undoubtedly 
responsible  for  many  crown  galls. 

CAUSAL  ORGANISM. — Pseudomonas  tumefaciens  is  a  short  rod  with  rounded  ends, 
motile  by  1-3  polar  flagella;  measures  1.2  to  2.5/1  by  0.5  to  o.Sju;  neither  spores 
nor  capsules  demonstrated;  pseudozoogloeae  occur;  involution  forms  present; 
stains  with  the  usual  anilin  stains;  Gram-negative;  on  agar,  slow,  four  to  six  days 
at  25°;  filiform,  raised,  white,  glistening,  somewhat  slimy;  potato,  growth  rapid, 
white,  smooth,  wet-glistening;  gelatin  stab,  filiform,  no  liquefaction;  moderate,  flat, 
filiform,  white,  smooth,  glistening,  no  liquefaction;  blood  serum,  moderate;  broth,  ring 
or  pellicle,  clouding  absent  or  inconspicuous;  milk,  coagulation  delayed,  curd  not 
peptonized,  litmus  gradually  blued  then  reduced;  silicate  jelly,  slow  white  growth; 
Cohn's  solution,  scanty  or  absent;  Uschinsky's  solution,  scanty,  not  viscid;  NaCl 
bouillon,  4  per  cent  inhibits,  3  per  cent  retards;  bouillon  over  chloroform,  growth 
unrestrained;  no  gas  from  sugars;  ammonia  is  produced^  nitrates  not  reduced;  indol 
production  small;  thermal  death-point  51°;  optimum  reaction  between  +14  and 
+24  Fuller's  scale;  opt.  temp.  25°  to  28°,  max.  37°,  min.  positive  at  o°;  killed 
readily  by  drying;  moderately  sensitive  to  sunlight;  invertase  and  rennet  thought 
to  be  produced. 

CONTROL. — Thorough  inspection  of  nursery  stock  and  care  in  the 
cultivation  of  orchards  "hot  to  wound  the  crowns  are  important  factors. 

*  Smith,  Erw.  P.,  Brown,  Nellie  A.,  McCulloch,  Lucia,  "The  Structure  and  Development  of 
Crown  Gall;  A  Plant  Cancer.  Bull.  255,  Bur.  Plant  Ind.  U.  S.  Dept.  Agric.,  1912. 


GALLS   AND   TUMORS  969 

Plant  on  uninfected  land  and  avoid  heeling  in  healthy  stock  into  soil 
that  has  previously  borne  diseased  plants. 

Removing  the  galls  results  in  no  practical  benefit. 

OLIVE  KNOT 
Bacterium   savastanoi — Smith*  f 

The  olive  knot  has  been  known  for  many  years,  and  is  even  des- 
cribed by  the  early  Roman  writers;  its  bacterial  nature,  however,  has 
been  recognized  only  since  1886.  It  is  most  prevalent  in  those  countries 
which  border  on  the  Mediterranean  Sea,  but  it  also  occurs  in  the  olive 
growing  sections  of  California. 

So  far  as  is  known,  the  causal  organism  enters  the  twigs  and  leaves 
of  the  olive  through  wounds,  and  there  produces  roughened,  wart-like 
swellings.  The  growth  of  the  knots  usually  begins  in  the  spring,  and 
later  in  the  season,  if  the  trees  are  badly  diseased,  they  show  scant 
foliage,  limited  growth,  and  occasionally  dead  branches,  especially 
where  the  galls  have  entirely  encircled  the  twigs. 

"FINGERS  AND  TOES"  OR  "STUMP  ROOT"  OF  CABBAGESJ 
Plasmodiophora  brassicce — Woronin  (1877) 

This  organism  which  is  classified  as  a  rhizopod  by  many  is  the 
cause  of  a  common  disease  of  the  roots  of  cabbages  and  of  other  crucif- 
erous plants.  The  disease  is  sometimes  called  " fingers  and  toes." 
It  may  cause  much  damage  in  market  gardens.  In  it  the  roots  are 
greatly  hyper trophied.  They  are  distorted  and  lumpy,  like  fingers 
bent  and  swollen  with  rheumatism.  The  disease  may  be  controlled 
to  some  extent  through  the  destruction,  by  burning,  of  all  infected 
material  as  soon  as  the  disease  is  recognized. 

It  is  usually  considered  well  to  rely  on  the  rotation  of  crops  or,  in 
case  the  soil  has  become  generally  infested,  to  plant  crops  of  another 
type  for  several  years  in  order  to  prevent  losses  from  this  infection. 
The  plants  attacked  are  recognized  by  their  stunted  appearance  and  by 

*  Smith,  Erw.,  Bull.  131,  Part  IV    Bur  of  Plant  Industry,  U.  S.  Dept.  of  Agriculture,  1908. 

t  Savastano,  L.  Les  maladies  de  J'olivier  et  la  tuberculose  en  particulier.  Comp.  Rend.  103, 
1144,  1116.  II  bacillo  della  tuberculosi  dell'olivo,  nota  suppletiva.  Rend.  Lincei  5:92-94. 
1889. 

J  Prepared  by  J.  L.  Todd. 


970 


MICROBIAL  DISEASES    OF   PLANTS 


the  tendency  of  the  leaves  to  wilt  or  turn  yellow.  A  microscopial 
examination  of  the  roots  will  reveal  the  distinctive  protozoa  which 
cause  the  disease. 

The  spores  are  liberated  with  the  disintegration  of  the  diseased  roots 
and  become  disseminated  in  the  soil  during  cultivation.  Under  ap- 
propriate conditions  the  spore  is  ruptured  and  a  small  flagellated, 
amoeboid  organism  emerges.  It  is  in  this  form  that  the  parasites 


FIG.  199. — Roots  of   Cabbage  plant  showing  characteristic  hypertrophy  due   to 
Plasmodiophora  brassica.     (Woronin.') 

penetrate  the  roots  of  the  young  plants  in  which  they  complete  their 
development.  The  youngest  forms  seen  within  the  vegetable  cells 
possess  two  nuclei  each  with  a  central  mass  of  chroma  tin  or  karyosome. 
Several  organisms  frequently  invade  a  single  cell.  As  they  grow,  there 
is  a  multiplication  of  nuclei  and  the  associated  organisms  tend  to  fuse 
together  to  form  plasmodia.  Subsequently  there  occurs  a  series  of 
changes.  Some  of  these  changes  are  readily  distinguished;  but  others 
are  more  difficult  to  follow.  The  nuclei  first  lose  the  greater  part 


GALLS   AND   TUMORS 


971 


of  their  chromatin  and  appear  pale  and  indistinct,  while  attraction 
spheres  appear  at  opposite  poles.  The  nuclei  then  divide  twice  by 
karyokinesis  and  a  small  amount  of  cytoplasm  is  separated  off,  con- 
stituting a  gamete.  The  gametes  now  unite  in  pairs  and  each  pair 
becomes  encysted  to  form  a  spore. 


FIG.  200. — Plasmodiophora  brassica.  A,  A  plant  cell  filled  with  parasites  the 
nuclei  of  which  are  undergoing  mitotic  division  (at  the  top  is  the  nucleus  of  the 
plant  cell).  B,  Two  plant  cells  with  developed  and  partly  developed  spores.  (After 
Prowazek,  from  Doflein.) 


Whether  during  the  multiplication  of  these  organisms  in  the  plant, 
they  are  able  to  migrate  to  other  cells  and  thus  spread  the  infection  has 
been  questioned.  A  number  of  investigators  believe  that  the  number 
of  infested  cells  is  only  increased  by  the  division  of  the  infected  plant 
cells  which  not  only  are  greatly  enlarged  but  also  show  evidence  of 


972  MICROBIAL  DISEASES    OF   PLANTS 

proliferation  in  the  presence  of  dividing  cells.  The  hypertrophy  of 
the  plant  cell  is  associated  with  hypertrophy  of  its  nucleus  and  it  is 
evident  that  the  growth  and  increase  of  the  parasite  is  favored  by 
the  reaction  which  its  presence  excites. 

TUBERCULOSIS  or  SUGAR-BEET 
Pseudomonas  beticola — Smith 

HISTORY. — This  new  disease  of  the  sugar-beet,  resembling  somewhat 
crown  gall  on  the  surface,  but  distinct  from  it,  was  first  observed  in 
the  autumn  of  1910  on  beets  from  Colorado  and  Kansas. 

SYMPTOMS. — Affected  beets  bear  numerous  wart-like  outgrowths  or 
tubercles  on  the  upper  portion  of  the  root.  On  section  these  show 
small,  water-soaked,  brownish  areas  with  more  or  less  necrotic  tissue 
in  their  interiors;  such  areas  may  develop  small  central  cavities,  and 
the  softening  may  extend  into  the  ungalled  part  of  the  beet;  the  dis- 
eased parts  appear  mucilaginous  and  stringy  when  touched,  and  under 
the  microscope  this  broken-down  tissue  is  found  to  be  swarming  with 
bacteria. 

CAUSAL  ORGANISM. — According  to  Smith*  Pseudomonas  beticola,  n.  sp.,  is  a 
motile  rod  with  rounded  ends,  single  or  in  pairs,  chains  or  clumps;  measures  0.6 
to  0.8  by  1.5  to  2.o/i;  flagella  polar;  no  spores  observed;  capsule  present;  liquefies 
gelatin,  but  not  blood  serum;  grows  in  beef  bouillon  containing  9  per  cent  NaCl; 
uniform  clouding  and  copious  pellicle  which  falls  easily  in  bouillon;  thermal  death- 
point  51°;  grows  at  37°  but  best  at  20°;  grows  slowly  at  i°;  produces  a  yellow 
rim  and  pellicle  in  plain  milk  which  is  slowly  coagulated;  whey  separates  slowly; 
litmus  mUk  is  blued  and  later  reduced;  grows  readily  in  Uschinsky's  solution, 
viscid;  no  growth  in  Cohn's  solution;  moderate  growth  on  potato;  does  not 
produce  gas  from  dextrose,  lactose,  saccharose,  maltose,  mannite  or  glycerin;  agar 
colonies,  circular,  smooth  or  wrinkled;  indol  is  produced;  grows  in  bouillon  over 
chloroform;  resists  drying;  stains  by  Gram;  is  yellow  or  becomes  yellow  on  all 
ordinary  media. 

*  Smith,  Erwin  P.,  "Crown  Gall  of  Plants:  Its  Cause  and  Remedy."  Bull.  213,  Bur.  Plant 
Ind.,  U.  S.  Dept.,  Agr.,  p.  194,  1911. 


CHAPTER  III 

LEAF  SPOTS 

CITRUS  CANKER 

Pseudomonas  citri — Hasse 

The  disease  was  probably  introduced  into  the  United  States  on 
nursery  stock  from  Japan,  and  since  1912,  has  occurred  in  Florida, 
Alabama,  Mississippi,  Louisiana,  and  Texas. 

SYMPTOMS. — According  to  Stevens,*  citrus  canker  attacks  all 
varieties  of  citrus  trees  of  any  commercial  value  in  Florida,  but  it  is 
most  severe  on  the  grape  fruit.  Under  field  conditions  a  characteristic 
spotting  of  the  fruit,  foliage  and  twigs  is  produced  which  appears  as 
small  light-brown  spots,  1.5  mm.-6  mm.  (J{6  to  J£  inch)  in  diameter. 
These  spots  may  occur  singly  or  several  may  coalesce  to  form  an  irregular 
area;  they  are  raised  above  the  adjoining  tissue  and  are  made  up  of  a 
spongy  mass  of  dead  cells,  covered  by  a  thin  white  or  grayish  membrane, 
which  ultimately  ruptures  forming  a  ragged  margin  around  the  spot. 
The  fruit  is  especially  susceptible  to  the  infection,  and  drops  soon  after  it 
is  attacked.  The  disease  is  spread  rapidly  from  one  part  of  the  tree  to 
another  by  insects,  rains  and  heavy  dews,  so  that  when  once  infected,  a 
tree  frequently  becomes  worthless  in  two  or  three  months. 

CAUSAL  ORGANISM. — Miss  Clara  H.  Hasse  f  has  described  the  causal  organism, 
Ps.  citri,  as  a  short  rod  with  rounded  ends,  motile  by  a  single  polar  flagellum. 

On  nutrient  agar,  the  growth  is  filiform,  shining,  dull  yellow  in  color;  on  potato, 
bright  yellow,  shining,  viscid.  In  nutrient  broth,  a  yellow  ring  is  formed  at  the 
surface  in  old  cultures.  Litmus  milk  becomes  deeper  blue,  and  the  casein  is  pre- 
cipitated. Gelatin  is  liquefied.  Indol  is  not  produced.  No  gas  is  formed  from 
sugars  in  Dunham's  solution.  Growth  is  slight  in  Uschinsky's  solution,  and  nitrates 
are  not  reduced  in  starch  nitrate  solution.  The  organism  grows  best  under  aerobic 
conditions. 

*  Stevens,  H.  E.,  "Citrus  Canker,  I,  II,  III,"  Bulls.  122, 124,  128,  Fla.  Exp.  Sta.,  1914,  1915. 
f  Hasse,  Clara  H.,  "Pseudomonas  citri,  the  Cause  of  Citrus  Canker,"  Jour.  A«r.  Res.,  Vol. 
IV,  No.  I,  p.  97,  1915. 

973 


974  MICROBIAL  DISEASES    OF   PLANTS 

METHOD  OF  INFECTION. — Experimental  evidence  goes  to  show  that 
infection  takes  place  through  stomata  as  well  as  through  wounds 
produced  by  insects,  or  by  other  mechanical  injuries. 

PATHOGENESIS. — According  to  Berger,  the  following  citrus  varieties 
are  subject  to  citrus  canker:  Pomelo,  citrus  trifoliata,  wild  lime,  Navel, 
sweet  seedlings,  Satsuma,  tangerine,  King  orange  and  lemon. 

CONTROL. — Removal  of  the  affected  parts  of  the  tree  by  pruning  has 
proven  a  complete  failure  as  a  control  measure,  and  the  only  practical 
means  of  handling  the  disease  appears  to  be  the  prompt  and  complete 
destruction,  by  burning,  of  all  stock  that  shows  the  slightest  trace  of 
infection. 

ANGULAR  LEAF-SPOT  OF  CUCUMBERS 
Pseudomonas  lachrymans — Erw.  Smith  and  Bryan 

HISTORY  AND  DISTRIBUTION. — The  angular  leaf-spot  of  cucumbers  is 
a  widespread  disease  occurring  in  many  of  the  Eastern  and  Middle 
Western  States.  It  has  been  recognized  in  the  field  for  more  than 
twenty  years,  but  it  was  not  until  1914  that  the  causal  organism  was 
isolated. 

SYMPTOMS. — The  disease  is  characterized  by  the  "numerous,  often 
confluent,  angular,  dry,  brown  spots  which  tear  or  drop  out  when  dry, 
giving  to  the  leaves  a  ragged  appearance.  In  the  early  stages  a  bac- 
terial exudate  collects  in  drops  on  the  lower  surface  during,  the  night 
and  dries  whitish,"*  and  because  of  these  tear-like  drops  of  exudate  the 
specific  name  lachrymans  has  been  suggested  for  the  causal  organism. 
The  young  stems  and  petioles  may  become  soft-rotted  and  crack  open, 
but  there  is  little  evidence  that  the  fruit  itself  suffers  from  the  disease, 
other  than  indirectly  from  lack  of  nourishment  resulting  from  the 
destruction  of  the  active  leaf  surface. 

CAUSAL  ORGANISM. — Pseudomonas  lachrymans  is  a  short  rod  with  rounded 
ends,  motile  by  means  of  1-5  polar  flagella.  No  spores  have  been  observed;  capsules 
are  formed  on  agar  and  in  milk.  It  is  Gram-negative  and  is  not  acid  fast. 

On  agar,  the  growth  is  smooth,  shining,  transparent,  white;  agar  colonies, 
two  to  four  days  old,  exhibit  opaque  white  centers  which  spread  in  radiating  lines 
into  the  thin  margin.  Gelatin  is  liquefied  slowly,  and  as  the  liquefaction  progresses 
the  upper  part  becomes  stratiform,  the  lower  part  bluntly  funnel-shaped.  In 

*  Smith,  Erw.  F.  and  Bryan,  Mary  Katherine;  "Angular  Leaf-spot  of  Cucumbers,"  Jour. 
Agr.  Res.,  Vol.  V,  No.  n,  pp.  465,  475,  1915. 


LEAF    SPOTS 


975 


nutrient  broth  moderate  clouding  occurs,  and  a  membranous  pellicle  is  formed 
which  breaks  readily  on  shaking.  On  potato,  the  growth  is  slimy,  shining,  creamy- 
white.  Plain  milk  clears  slowly  without  coagulation,  becoming  translucent  and 
tawny-olive  with  age.  Lavender-colored  litmus  milk  is  completely  blued  in  three 
days,  and  a  creamy- white  pellicle  is  formed  at  the  surface;  clearing  is  complete  in 
twenty  days,  and  later  the  blue  color  bleaches  out  leaving  the  fluid  a  translucent 
brown. 

The  organism  grows  in  Uschinsky's,  Fermi's,  and  Cohn's  solutions  producing  a 
green  coloration  in  the  first  two. 

No  gas  is  formed  from  the  ordinary  sugars;  acid  is  produced  from  saccharose 
and  dextrose.  Nitrates  are  not  reduced.  Hydrogen  sulphid  is  not  formed.  A 
small  amount  of  indol  is  produced  in  2  per  cent  peptone  water  and  peptonized 
Uschinsky's  solution.  Methylene  blue  in  milk  is  rapidly  reduced.  The  organism 
is  an  obligate  aerobe. 

Optimum  temperature  is  25°  to  27°.;  no  growth  at  36°. 

METHOD  OF  INFECTION. — The  causal  organism  enters  the  leaves 
through  the  stomata,  no  wounds  being  necessary. 

CONTROL. — Laboratory  experiments  upon  the  germicidal  action  of 
copper  sulphate  on  Ps.  lachrymans  suggest  that  Bordeaux  mixture, 
properly  applied,  may  be  a  remedy  for  the  disease. 

SPOT  OF  THE  LARKSPUR 
Bacillus  delphini — Erw.  Smith 

So  far  as  is  known,  this  disease  occurs  only  on  the  larkspurs  of 
Massachusetts.  Infection  takes  place  through  the  stomata,  resulting 
in  numerous  black  spots  on  the  leaves  and  stems. 

CAUSAL  ORGANISM. — Smith*  describes  the  organism  as  a  motile,  gray- white, 
non-liquefying,  nitrate  reducing  bacillus.  Agar  colony  has  characteristic  wrinkled 
structure.  Grows  in  Uschinsky's  solution.  No  growth  at  37°;  thermal  death- 
point  48°  to  49.1°. 

BACTERIAL  SPOT  OF  PLUM  AND  PEACH 
Pseudomonas  pruni — Erw.  Smith 

The  first  occurrence  of  the  bacterial  spot  was  reported  on  the  Japan- 
ese plum  in  Michigan,  f  Later,  what  appeared  to  be  the  same  disease 
was  observed  on  the  peach  in  Georgia^  and  Connecticut,  and  more 
recently  it  has  been  found  throughout  the  South  and  Middle  West. 

*  Smith,  Erw.  P.,  Science,  N.  S.,  Vol.  XIX,  No.  480,  p.  418,  1904. 
t  Smith,  Erw.,  Science,  N.  S.,  Vol.  XVIII,  429,  p.  456,  1903. 
j  Rorer,  J.  B.,  Science,  N.  S.,  Vol.  XXIX,  7S3.  p.  914.  1909- 


976  MICROBIAL   DISEASES    OF   PLANTS 

SYMPTOMS. — On  the  plum,  the  leaves  and  green  fruit  exhibit  numer- 
ous small,  water-soaked  spots;  later  the  diseased  tissue  of  the  leaves 
falls  out,  giving  a  shot-hole  appearance,  and  the  plums  show  black, 
sunken  areas  and  deep  cracks.  The  spots  may  reach  a  diameter  of 
one-fourth  to  one-half  inch. 

On  the  peach  leaves,  angular,  purplish-brown  spots  one-eighth  to 
one-fourth  inch  in  diameter  are  formed,  which  drop  out  giving  the  shot- 
hole  effect.  The  organism  also  attacks  the  young  twigs  and  fruit.  It 
destroys  the  bark  of  the  former,  producing  black,  sunken  areas,  while 
on  the  latter  it  causes  small  purplish  spots  over  which  the  skin  cracks. 

In  both  the  plum  and  the  peach,  infection  is  believed  to  take  place 
through  the  stomata.  It  is  primarily  a  disease  of  the  parenchyma, 
but  the  vascular  system  is  invaded  ultimately. 

CAUSAL  ORGANISM. — Ps.  pruni  Smith,  is  a  small  rod,  motile  by  one  to  several 
polar  flagella.  It  grows  readily  upon  the  ordinary  culture  media.  On  agar,  it 
resembles  Ps.  campestris,  producing  a  distinctly  yellow  pigment,  but  is  distinguished 
by  its  feeble  growth  on  potato  and  by  its  growth  in  Uschinsky's  solution,  which  is 
converted  into  a  viscid  material  like  egg  albumin.  Gelatin  liquefied  slowly.  Casein 
of  milk  precipitated  slowly  and  redissolved;  litmus  reduced  but  color  restored  later. 
No  gas  produced.  Thermal  death-point  51°. 

DISEASE  OF  SUGAR-BEET  AND  NASTURTIUM  LEAVES 
Pseudomonas  aptatum — Brown  and  Jamieson 

HISTORY. — The  bacterial  leaf  spot  of  sugar-beet  and  nasturtium 
leaves  was  first  observed  in  the  summer  and  spring  of  1908  on  nastur- 
tium leaves  growing  near  Richmond,  Va.,  and  on  sugar-beet  leaves 
obtained  from  Garland,  Utah;  more  recently  the  trouble  has  been  noted 
in  California  and  Oregon  on  the  sugar-beet. 

SYMPTOMS. — Affected  nasturtium  leaves  exhibit  water-soaked  and 
brownish  spots  from  2  to  5  mm.  in  diameter.  The  sugar-beet  leaves 
disclose  "dark-brown,  often  black,  irregular  spots  and  streaks  from 
3  mm.  to  15  mm.  in  diameter.  They  occur  on  the  petiole,  midrib,  and 
larger  veins."  Occasionally  the  discoloration  extends  along  the  veins, 
and  the  tissue  on  either  side  is  brown  and  dry;  sometimes  cork-like 
protuberances  occur  at  the  central  point  of  the  spots.  In  badly  dis- 
eased petioles  the  tissue  softens  as  though  affected  with  a  soft  rot,  but 
where  the  infection  is  mild  there  is  no  indication  of  this  condition. 


LEAF   SPOTS  977 

Microscopic  examination  of  the  diseased  spots  and  adjacent  area  shows 
the  tissue  to  be  filled  with  a  large  number  of  active  bacteria.  Sections 
cut  from  the  central  portions  of  the  diseased  areas  show  the  cell  walls 
to  be  ruptured  or  collapsed,  while  the  cells  bordering  the  ruptured 
places  show  that  the  bacteria  are  in  the  cells.  The  disease  is  repro- 
duced readily  with  typical  symptoms  by  means  of  needle  prick  inocula- 
tions with  pure  cultures.  So  far  as  has  been  observed,  the  causal 
organism  does  not  attack  the  beet  root,  but  is  confined  strictly  to  the 
beet  leaf. 

CAUSAL  ORGANISM. — According  to  Brown  and  Jarmeson,* Pseudomonas  aptatum, 
n.  sp.,  is  a  short,  motile  rod  with  rounded  ends;  flagella,  bi-polar;  involution  forms 
.rare;  no  spores  or  capsules  observed;  pseudozoogloeae  occur;  aerobic;  smooth  whitish 
colonies  on  agar  plate  with  fish  scale-like  markings;  clouds  beef  bouillon  in  eighteen 
to  twenty-four  hours;  produces  alkaline  reaction  in  litmus  milk,  with  a  gradual 
separation  of  whey  from  curd;  liquefies  gelatin;  produces  ammonia;  no  reduction 
of  nitrates;  fluorescence  greenish;  no  diastasic  action  on  potato  starch;  grows  in 
Uschinsky's  and  Fermi's  solutions;  indol  produced  after  ten  days;  optimum 
temperature  27°  to  28°;  maximum  34°  to  35°;  minimum  i°;  thermal  death- 
point  47.5°  to  48°;  vitality  four  to  ten  months  in  beef  agar,  ten  to  twelve  months 
in  beef  bouillon,  depending  on  temperature;  growth  good  on  litmus-lactose  agar; 
growth  much  retarded  on  gentian  violet  agar;  stains  readily  with  basic  anilin  dyes; 
not  acid  fast;  not  stained  by  Gram;  tolerates  acids;  oxalic  o.i  per  cent;  tartaric 
0.2  per  cent;  hydrochloric  o.i  per  cent;  tolerates  sodium  hydroxide  in  beef 
bouillon,  — 18  Fuller's  scale;  no  growth  in  Cohn's  solution;  killed  readily  by  drying; 
not  very  sensitive  to  sunlight;  retains  its  virulence  two  to  three  years. 

PATHOGENESIS. — Pathogenic  to  nasturtium  and  sugar-beet  leaves; 
spots  have  been  produced  by  artificial  inoculations  on  leaves  of  pepper, 
lettuce,  egg  plant,  and  upon  the  leaves  and  pods  of  the  bean  plant. 

METHOD  OF  INFECTION. — It  is  believed  that  infection  takes  place 
only  in  bruised  or  wounded  tissue,  due  to  insects  or  to  mechanical  injury. 

CONTROL. — No  practical  methods  of  control  have  been  undertaken. 

•  Brown.  Nellie  A.,  Jamieson,  Clara  O..  "A  Bacterium  Causing  a  Disease  of  Sugar-beet  and 
Nasturtium  Leaves,"  Jour.  Agr.  Res.,  Vol.  I,  No.  3,  P-  189,  1913- 


CHAPTER  IV 

ROTS 

BLACK  ROT  OF  CABBAGE 
Pseudomonas  campestris — Pammel  (Erw.  Smith) 

This  disease  is  widely  distributed  in  the  United  States  and  Europe 
and  has  become  so  serious  on  many  truck  farms  that  gardeners  dread 
its  appearance  as  much  as  orchardists  do  pear  blight.  It  is  not  confined 
to  cabbage,  but  it  attacks  other  cruciferous  plants  such  as  cauliflower 
kohlrabi,  kale,  rape,  turnips,  mangels,  rutabagas  and  mustards. 

SYMPTOMS. — The  first  symptom  is  the  withered,  yellow  margin  of 
the  leaf,  giving  the  impression  of  a  "burned  edge."  The  progress  of 
the  disease  is  inward  and  downward  through  the  vascular  system,  as 
is  indicated  by  the  brown  or  black  color  of  the  veins  and  midrib.  The 
tissue  of  the  vascular  bundles  is  destroyed  and  the  cell  walls  of  the 
adjacent  tissue  are  dissolved,  presumably  by  a  cytolytic  enzyme.*  In 
this  way  practically  all  of  the  tissues  are  softened,  disorganized,  and  a 
general  infection  of  the  whole  plant  may  follow.  Diseased  leaves  fall 
prematurely,  leaving  a  long  naked  stalk  with  a  tuft  of  leaves  at  the  top. 
The  dwarfed,  one-sided  growth  of  the  heads,  and  in  some  cases  the 
failure  to  produce  heads  is  characteristic. 

METHOD  OF  INFECTION. — Water  poref  infection  along  the  margin 
of  the  leaf  is  believed  to  be  the  most  common  method  of  entrance, 
although  root  inoculation  at  the  time  of  transplanting  undoubtedly 
takes  place  also.  It  has  been  shown,  further,  that  the  germ  is  intro- 
duced on  the  seed-t 

CAUSAL  ORGANISMS.  § — Pseudomonas  campestris  Pammel,  is  a  short  rod  with 
rounded  ends,  relatively  shorter  in  the  host  tissue  than  on  culture  media,  0.7/1  to 

*  Smith,  E.  F. :  Bull.  25,  Bur.  Plant  Industry,  U.  S.  Dept.  Agr.,  1903. 

t  Russell,  E.  J.:  Bull.  65,  Wisconsin  Exp.  Station,  1898.  Smith,  E.  P.:  Farmers'  Bull.  63, 
U.  S.  Dept.  Agriculture,  1898. 

t  Harding,  H.  A.:  Bull.  251,  N.  Y.  Experiment  Station,  1904. 

§  For  a  means  of  distinguishing  Ps.  campestris,  Ps.  phaseoli,  Ps.  hyacinthi  and  Ps.  stewarti. 
the  student  is  referred  to  Bull.  28,  p.  149,  Div.  Veg.  Phys.  and  Path.,  U.  S.  Dept.  Agr.,  1901. 

978 


ROTS  979 

3-oji  by  0.4/i  to  0.5^;  motile  when  young  by  one  polar  flagellum;  no  capsule  dem- 
onstrated and  no  spores  observed;  zcoglceae  in  liquid  cultures.  Stains  readily  with 
aqueous  stains.  Gram-negative. 

It  grows  readily  in  the  ordinary  culture  media.  Upon  potato,  growth  is  charac- 
teristic; at  first  light  yellow,  and  in  old  cultures  a  golden  brown,  abundant,  moist, 
shining,  slimy.  Gelatin  liquefied  slowly.  Litmus  milk  becomes  slightly  alkaline, 
casein  separated  and  gradually  redissolved.  On  nutrient  agar,  translucent,  yellow 
slime.  No  gas  from  dextrose,  lactose,  etc.  Uschinsky's  solution,  growth  retarded 
and  feeble.  Aerobic.  Indol  produced.  Nitrates  not  reduced.  Diastase  produced. 
Optimum  temperature,  25°  to  30°;  thermal  death-point,  51.5°. 

CONTROL. — The  removal  of  diseased  leaves  in  the  early  stages  has 
been  practiced  by  some  growers  with  success,  but  care  must  be  taken 
not  to  remove  so  many  that  growth  will  be  checked.  Manure  con- 
taining diseased  cabbage  refuse  must  not  be  used.  Seed  disinfection 
with  1:1,000  mercuric  chloride,  fifteen  minutes,  or  formalin  1:200, 
twenty  minutes,  is  recommended.  Rotation  of  crops,  and  planting  on 
new  land  should  be  practised  whenever  possible.  If  practicable,  the 
seed  bed  should  be  made  in  sterilized  soil,  so  that  the  plants  will  be 
healthy  when  set  in  the  field. 


WAKKER'S  HYACINTH  DISEASE 
Pseudomonas  hyacinthi — Wakker 

HISTORY. — One  of  the  earliest  landmarks  in  the  study  of  bacterial 
diseases  of  plants  is  the  excellent  contribution  of  Dr.  J.  H.  Wakker,*  a 
Dutch  botanist,  who  between  1883  and  1888  published  five  papers  on  a 
disease  of  the  hyacinth,  caused  by  Ps.  hyacinthi.  Erwin  F.  Smith  f  has 
carried  the  investigation  farther  and  has  described  the  causal  organism 
more  fully.  The  disease  was  first  observed  in  the  Netherlands  where  it 
frequently  causes  serious  losses  in  the  hyacinth  gardens.  It  is  not 
known  to  occur  in  any  other  part  of  the  world. 

SYMPTOMS. — The  disease  is  characterized  by  a  yellow  striping  of 
the  green  leaves  and  the  bright  yellow  slime  produced  in  the  vascular 
bundles  of  the  bulb.  The  infection  in  the  leaf  spreads  slowly  to  the 
bulb  by  the  multiplication  of  bacteria  in  the  vascular  system,  filling  the 

*  Wakker,  J.  H.:  Bot.  Centralbl.,  18831  M,  P-  315;  Archives  neerlandaises  des  sci.  ex.  et  natu- 
relles.  TomelXXIII.  pp.  18-20. 

t  Smith,  Erwin  P.,  "Wakker's  Hyacinth  Germ,"  Bull.  No.  26,  U.  S.  Dept.  Agr.,  Dir.  Veg. 
Phys.  and  Path.,  1901. 


980  MICROBIAL  DISEASES  OF  PLANTS 

vessels,  especially  those  of  the  bulb,  with  a  bright  yellow  bacterial  slime. 
In  time,  the  walls  of  the  vessels  are  destroyed  and  large  cavities  are 
formed  in  the  fibro-vascular  bundles.  The  disease  does  not  spread 
rapidly  from  bundle  to  bundle  in  the  bulb,  but  is  confined  for  a  long 
time  to  the  vessels  first  involved,  a  year  or  more  being  required  for  the 
destruction  of  the  host  plant.  This  is  due,  largely,  to  the  resistance 
offered  by  the  cells  of  the  parenchyma  to  bacterial  invasion. 

METHOD  OF  INFECTION. — The  causal  organism  enters  through 
wounds  in  the  leaves  and  through  the  blossoms,  and  when  the  disease  is 
once  established,  it  is  probably  spread  by  insects  which  visit  the  blos- 
soms or  eat  the  leaves.  Daughter  bulbs  contract  the  infection  from 
mother  bulbs.  Wakker  believed  the  disease  to  be  transmitted  often 
by  knives  used  around  sick  plants. 

CAUSAL  ORGANISM. — Pseudomonas  hyacinthi  Wakker,  according  to  Erwin  F. 
Smith,  is  a  medium-sized  rod  with  rounded  ends,  i.o/z  to  2.o/x  by  0.5^  to  0.7^,  motile 
by  one  polar  flagellum;  non-spore  forming. 

It  grows  well  upon  the  ordinary  culture  media,  on  most  of  which,  as  well  as  in 
the  host  plant,  it  produces  a  bright,  chrome-yellow  pigment.  Gelatin  and  blood 
serum  are  liquefied  slowly  (six  to  seven  days).  Milk  is  rendered  alkaline,  and  the 
casein  is  slowly  precipitated.  On  nutrient  agar,  growth  is  copious,  yellow,  smooth, 
wet-shining,  translucent,  spreading.  On  20  per  cent  cane  agar,  the  zooglcea  formed 
gives  the  growth  a  papillose,  verrucose  appearance.  Acid  but  no  gas  is  formed  in 
dextrose  and  saccharose  broth;  indol  produced  slowly.  Nitrates  not  reduced. 
Feeble  growth  in  Uschinsky's  solution.  Does  not  grow  at  37°;  optimum  tem- 
perature 28°  to  30°;  thermal  death-point  47.5°. 

The  hyacinth  is  the  only  known  host  plant. 

CONTROL. — Diseased  bulbs  should  be  removed  from  the  fields  and 
destroyed;  land  on  which  the  disease  is  present  should  be  used  for  other 
crops;  the  use  of  infected  tools  without  thorough  disinfection  should  be 
avoided.  The  selection  and  breeding  of  disease  resistant  varieties,  as 
advised  by  Wakker,  suggests  the  most  practical  way  of  controlling  the 
trouble. 

BLACK  LEG  OR  BASAL  STEM  ROT  OF  POTATO 
Bacillus  phytophthorus — Appel* 

The  disease  is  prevalent  in  the  United  States  and  Europe.  It 
appears  to  originate  in  the  seed  tubers  from  which  it  extends  upward 

*  Appel,  Otto,  "Untersuchungen  u.  d.  Schwarzbeinigkeit."  Arb.  Bio.  K.  G.  Amt.,  Berlin, 
1903. 


ROTS  981 

into  the  base  of  the  stem  causing  it  to  turn  black  and  rot.  The  vines 
grow  spindling,  turn  yellow  and  die  prematurely.  The  diseased  tubers 
may  rot  in  the  soil  or  later  when  in  storage  cause  a  soft  rot  of  the  crop. 

CAUSAL  ORGANISM. — Erwin  Smith  describes  the  causal  organism  as  a  non- 
spore  forming  bacillus,  motile  by  means  of  peritrichate  flagella.  It  stains  with  the 
ordinary  stains,  but  is  Gram-negative.  The  growth  is  grayish-white  on  agar  and 
on  gelatin  plates  large,  round,  white  colonies  develop  promptly.  Gelatin  is  liquefied 
with  funnel-shaped  liquefaction.  On  cooked  potato,  white  to  yellowish  growth. 
Raw  potato,  white  growth  and  black  stain.  There  is  a  slow  acid  coagulation  of 
milk  with  precipitation  of  casein  and  reduction  of  litmus.  Thick  pellicle  and 
heavy  precipitate  in  potato  juice.  No  growth  in  Cohn's  solution.  Moderate 
production  of  hydrogen  sulphide.  Nitrates  reduced.  No  indol.  Acid  from 
dextrose,  saccharose,  lactose,  maltose  and  galactose.  Some  gas  from  inosite, 
lactose  and  mannite.  Facultative  anaerobe.  Optimum  temperature,  28°  to  30°. 
Thermal  death-point,  47°. 

Closely  related  organisms  are  B.  solanisaprus  Harrison,  and  B.  atro- 
septicus  van  Hall. 

CONTROL. — In  view  of  the  fact  that  the  germs  are  introduced  with 
the  seed  potatoes,  thorough  disinfection  of  the  seed  with  formalin  is 
recommended. 

BUD-ROT  OF  THE  COCOANUT 

Bacillus  coli  (Escherich)  Migula 

HISTORY  AND  DISTRIBUTION. — The  bud-rot  of  the  cocoanut  has 
been  known  for  more  than  thirty  years  in  Cuba  and  is  to  be  found  dis- 
tributed more  or  less  generally  throughout  tropical  America  and  the 
eastern  tropics. 

SYMPTOMS. — Johnston*  states  that  in  the  acute  stages  of  the  disease, 
the  bud,  or  the  growing  point  in  the  center  of  the  crown,  is  affected  by 
a  vile-smelling  soft  rot  which  destroys  all  the  younger  tissues.  Most 
of  the  nuts  fall,  the  lower  leaves  turn  yellow  and  the  middle  folded  and 
undeveloped  leaves  die  and  hang  down  between  the  still  green  sur- 
rounding ones.  The  rot  gradually  spreads  from  the  base  of  one  spike  to 
another  until  all  are  involved  and  shed  their  nuts;  the  leaf  stalks  become 
so  rotten  at  their  bases  that  they  are  no  longer  able  to  maintain  their 
natural  position  and  droop  or  else  fall  off.  From  a  central  diseased  bud, 
the  infection  may  spread  downward  and  into  the  trunk  of  the  tree  for 

•  Johnston,  John  R.,  "The  History  and  Cause  of  Cocoanut  Bud  -rot,"  Bull.  228,  Bur.  Plant 
Ind..  U.  S.  Dept.  Agr.,  1912. 


982  MICROBIAL  DISEASES    OF   PLANTS 

a  short  distance,  rotting  out  the  fundamental  tissues  and  leaving  only 
the  fibers  which  are  too  hard  to  be  disintegrated. 

It  has  been  estimated  that  in  some  cocoanut  groves  from  75  to  90 
per  cent  of  the  trees  have  been  destroyed  by  the  rot. 

CAUSAL  ORGANISM. — B.  coli  (Escherich)  Migula. 

METHOD  OF  INFECTION. — It  is  believed  that  the  causal  organism 
enters  the  host  through  insect  bites  or  other  mechanical  injuries  to  the 
soft  tissue.  Insects,  birds  or  some  form  of  animal  life  are  held  respon- 
sible for  spreading  the  trouble. 

CONTROL. — The  removal  of  the  diseased  parts  of  a  tree  as  well  as 
spraying  have  proved  of  no  benefit  in  controlling  the  disease.  "The 
absolute  destruction  of  diseased  trees,  a  careful  watch  for  the  newly 
infected  cases,  and  their  immediate  removal  has  done  much  to  .prevent 
greater  loss  in  the  various  regions." 

BROWN  ROT,  A  LEAF-DISEASE  OF  TROPICAL  ORCHIDS 
Bacillus  cypripedii — S.  Hori 

HISTORY  AND  DISTRIBUTION. — The  brown  rot  of  orchids  was  first 
observed  by  Hori*  in  1906  on  orchids  growing  in  the  greenhouses  in 
Tokyo,  Japan.  Since  then  the  disease  has  been  noted  on  orchids  from 
Formosa  grown  in  their  natural  habitat  out  of  doors.  In  1898  v.  Peg- 
lionf  described  a  similar  trouble  in  Italy  which  may  be  identical  with 
the  above. 

SYMPTOMS. — The  rot  is  characterized  by  dirty  cinnamon  or  light 
umber  colored,  depressed  spots  on  the  leaf -blade;  these  become  darker 
with  age  and  may  increase  in  size  so  rapidly  that  the  entire  green  leaf 
is  discolored  (yellowish)  in  a  few  days  and  dies.  The  rotting  also 
spreads  downward  into  the  stem,  and  if  the  diseased  leaves  are  not 
removed  early,  the  entire  stalk  will  be  destroyed. 

CAUSAL  ORGANISM. — Bacillus  cypripedii  is  a  medium-sized  rod  with  rounded 
ends;  single  or  in  short  chains;  measures  1.5  to  zp  X  0.5  to  0.7/1;  stains  readily  with 
aniline  dyes;  Gram-positive;  motile  by  4  peritrichate  flagella;  non-spore  forming; 
smooth,  light  grayish  white  colony,  with  pearl  luster  on  agar;  dirty  cream  colony 

*Hori,  S.,  "A  Bacterial  Leaf-disease  of  Tropical  Orchids,"  Cent.  f.  Bakt.,  Abt.  II,  Bd.  31. 
p.  85,  1911. 

t  v.  Peglion,  "Bacteriosi  delle  folie  di  Oncidium  spec,"  Cent.  f.  Bakt.,  Abt.  II,  Bd.  5, 
P-  33,  1899. 


ROTS  983 

on  potato;  surface  film  on  bouillon;  liquefies  gelatin  rapidly;  coagulates  milk; 
ferments  glucose  with  production  of  H  and  COz  in  the  ratio  1:3;  indol  positive  after 
forty  days;  methylene  blue  reduced;  ammonia  and  H2S  produced  from  bouillon; 
enzymes:  amylase,  oxidase,  peroxidase;  facultative  anaerobe. 

PATHOGENESIS. — Pathogenic  to  orchids  grown  in  the  hothouse  and 
also  in  their  habitat. 

METHOD  OF  INFECTION. — The  germs  enter  the  leaf  tissue  chiefly 
through  wounds  caused  by  careless  washing. 

CONTROL. — Use  only  a  soft  sponge  soaked  in  a  i :  1000  solution  of 
mercuric  chloride  for  wiping  the  leaves,  and  avoid  excessive  watering 
as  this  favors  the  disease. 


ROT  OF  CAULIFLOWER  AND  ALLIED  PLANTS 
Bacillus  oleracea — Harrison 

HISTORY  AND  SYMPTOMS. — This  rot  of  cauliflower  and  allied  plants 
was  first  reported  in  1901  from  truck  gardens  in  the  vicinity  of  Guelph, 
Ontario.  It  is  characterized  by  a  soft  rot  of  the  roots  and  a  blackening 
of  the  stems  and  leaves.  Harrison*  has  found  this  condition  to  be 
traceable  to  an  actively  motile  bacillus  which  invades  the  intercellular 
spaces  of  the  plant  and  destroys  the  middle  lamellae. 

CAUSAL  ORGANISM. — Bacillus  o/eracec^-Harrison  is  a  rod  with  rounded  ends; 
occurs  single  or  in  short  chains;  measures  2  X  o.6/u;  motile  by  means  of  7  to  13 
peritrichate  flagella;  stains  with  the  ordinary  aniline  dyes;  Gram-negative;  in 
broth  heavy  turbidity  and  sediment,  no  pellicle;  stratiform  liquefaction  of  gelatin; 
on  agar  spreading,  thin,  whitish,  moist,  slightly  opalescent;  neutral  red  agar  no 
change  in  color;  litmus  milk  coagulated,  soft  curd  slowly  peptonized;  blood  serum 
slightly  liquefied;  growth  positive  in  Uschinsky's  and  Fermi's  solutions;  potato  waxy, 
straw-colored  to  moist,  shining;  opt.  temp.  30°,  max.  42°,  min.  5°;  thermal  death- 
point  55°;  facultative  anaerobe;  slight  reduction  of  nitrates;  indol  slight;  H^S 
positive;  slight  gas  from  glucose  and  lactose,  none  from  saccharose;  acid  from 
sugars;  enzymes:  proteolytic,  diastase,  cytase  (pectinase). 

METHOD  OF  INFECTION. — Infection  takes  place  chiefly  through 
wounds  due  either  to  mechanical  or  insect  injuries.  Warm  weather 
combined  with  excessive  moisture  appears  to  favor  the  spread  of  the 
disease. 

*  Harrison,  F.  C.,  "A  Bacterial  Disease  of  Cauliflower  (Brassica  oleracea)  and  Allied  Plants," 
Cent.  f.  Bakt..  Abt.  II.  Bd.  13,  PP-  46,  185,  1904. 


984  MICROBIAL  DISEASES    OF   PLANTS 

PATHOGENESIS. — Pathogenic  for  cauliflower,  cabbage,  and  turnips; 
a  soft  rot  can  be  produced  in  a  large  variety  of  vegetables  under  labora- 
tory conditions  by  pure  culture  inoculations. 

CONTROL. — Complete  destruction  of  diseased  crops  by  burning  and 
crop  rotation  are  to  be  recommended. 

Harding  and  Morse,*  from  their  extensive  comparative  studies  of 
microorganisms  producing  soft  rots  of  vegetables  make  Bacillus  oleracea 
of  Harrison  identical  with  B.  carotovorus  of  Jones. 

SOFT  ROT  OF  CALLA  LILY 
Bacillus  aroidece — Townsendf 

A  soft  rot  of  the  calla  lily,  distinct  from  other  soft  rots,  is  scattered 
over  the  calla-growing  sections  of  the  United  States.  The  disease  starts 
at  the  top  of  the  corm  and  causes  a  rotting  of  the  plant  at  or  just  below 
the  surface  of  the  ground.  As  a  result  the  leaves  and  flower  stalk  turn 
brown  and  fall  over.  The  healthy  corms  are  white,  but  the  infected 
ones  are  brown,  soft  and  watery. 

It  is  believed  that  the  causal  organism  lives  in  the  soil  and  enters  the 
plants  through  wounds.  The  disease  is  undoubtedly  spread  from  one 
locality  to  another  by  shipping  slightly  diseased  corms. 

As  a  means  of  control,  only  sound  corms  should  be  used,  and  the 
soil  in  the  calla  beds  should  be  changed  every  three  to  four  years. 

SOFT  ROT  OF  CARROT  AND  OTHER  VEGETABLES 
Bacillus  carotowrus — Jones 

A  number  of  the  cultivated  plants  of  the  north  temperate  zone, 
notably  those  grown  for  their  root  crops,  suffer,  at  times,  from  a  bac- 
terial rot  caused  by  a  liquefying  bacillus.  Although  probably  as  widely 
distributed  as  any  microorganism  parasitic  upon  plants,  it  was  not  de- 
scribed until  19014 

Bacillus  carotowrus  is  a  wound  parasite  which  invades  the  inter- 
cellular spaces,  dissolving  the  middle  lamellae  and  poi  tions  of  the  inner 

*  See  footnote,  p.  985. 

f  Townsend,  C.  O.,  Bull.  60,  Bur.  Plant  Ind..  U.  S.  Dept.  Agr.,  1904. 

t  Jones,  L.  R.,  "A  Soft  Rot  of  Carrot  and  Other  Vegetables,"  isth  Report  Vermont  Exp. 
Station,  p.  299,  1901. 


ROTS  985 

lamellae,  thereby  establishing  a  condition  which  is  known  as  a  soft  rot. 
Jones*  has  shown  this  solution  to  be  due  to  a  bacterial  enzyme  which 
he  has  named  pectinase. 

CAUSAL  ORGANISM. — The  organism  is  a  variable  rod,  majority  2.o/*  by  0.8/1 
rounded  ends,  motile  by  2  to  10  peritrichate  flagella;  no  endospores;  no  capsules; 
slight  pseudozooglceae.  Stains  readily  with  aqueous  stains.  Gram-negative. 

On  agar,  growth  abundant,  filiform  to  spreading,  glistening,  smooth,  white, 
opaque  to  opalescent.  Potato — glistening,  white,  decided  odor,  smooth,  butyrous, 
medium  grayed.  Gelatin  stab — filiform,  liquefaction  crateriform  to  infundi- 
buliform,  liquefaction  begins  second  day  and  complete  in  six  days.  Broth — thin 
pellicle,  clouding,  abundant  sediment.  Milk — coagulated,  slowly  peptonized, 
rendered  acid,  litmus  reduced.  Conn's  solution — no  growth.  Uschinsky's  solu- 
tion— abundant  growth.  Quick  tests;  soft  rot  of  uncooked  carrots,  turnips,  cab- 
bages. Slight  gas  produced  from  dextrose,  lactose,  saccharose,  but  not  glycerin. 
Acid  from  dextrose,  lactose,  saccharose  and  glycerin.  Nitrates  reduced.  Slight 
indol.  Thermal  death-point,  48°  to  50°;  grows  at  37°.  Optimum  temperature  25° 
to  30°.  Pathogenic  to  the  roots  of  carrot,  turnip,  rutabaga,  radish,  salsify,  parsnip, 
bulb  of  onion,  leaf  stalk  of  celery,  leaves  and  scapes  of  hyacinth,  cabbage,  cauli- 
flower, lettuce,  Irish  potato,  fruit  of  tomato,  eggplant  and  pepper. 

B.  oleracea  Harrison,  and  B.  omnivorus  van  Hall,  formerly  described  as  bacterial 
species  capable  of  producing  soft  rots,  have  been  reported  by  Harding  and  Morse  f 
as  identical  with  B.  carotovorus  and  therefore  to  be  recognized  no  longer  as  distinct 
species. 

CONTROL. — Jones  believes  that  the  soft  rots  can  be  practically  held 
in  check  by  rotation  of  crops;  by  not  using  manure  into  which  garden 
refuse  has  been  thrown;  by  drying  the  surface  of  the  roots  thoroughly 
and  exposing  them  to  bright  sunshine  before  storage;  by  maintaining  a 
constant  low  temperature  (4°)  during  storage. 


SOFT  ROT  or  HYACINTH 
Bacillus  hyacinthi  septicus — Heinzt 

A  very  active  soft  rot  of  the  hyacinth  bulb,  producing  a  bad  smelling, 
slimy  condition  in  a  few  days,  has  been  described  by  Heinz  as  caused 
by  an  unpigmented,  motile  bacillus. 

*  Jones,  L.  R.,  "Pectinase,  the  cytolytic  enzyme  produced  by  Bacillus  carotovorus  and 
certain  other  soft  rot  organisms."     Tech.  Bull.  II,  New  York  Agr.  Exp.  Sta.,  1909. 
t  Harding  and  Morse,  Tech.  Bull.  n.  New  York  Exp.  Sta.,  1909. 
j  Heinz,  Cent.  f.  Bakt.,  5,  P-  535,  1899. 


986  MICROBIAL  DISEASES    OF   PLANTS 

SOFT  ROT  OF  MUSKMELON 
Bacillus  melonis — Giddings 

HISTORY. — Toward  the  close  of  the  season  of  1907  the  muskmelons 
in  certain  sections  of  Vermont  were  attacked  by  a  soft  rot.  An 
investigation  of  the  cause  of  the  trouble  by  Giddings*  showed  it  to  be 
due  to  a  microorganism  which  he  has  called  B.  melonis. 

SYMPTOMS. — The  decay  usually  begins  on  that  part  of  the  melon 
next  to  the  soil  as  shown  by  the  shrunken  but  generally  unbroken  skin 
over  the  soft  diseased  area.  There  is  a  complete  collapse  of  the  melons 
accompanied  by  some  frothing  and  a  disagreeable  odor  in  the  last  stages. 
A  microscopic  examination  of  the  diseased  tissue,  both  fresh  and  killed, 
shows  that  the  bacterial  invasion  is  purely  intercellular,  and  the  patho- 
logical condition  of  the  tissue  manifested  as  a  soft  rot  is  due  to  the 
solution  of  the  middle  lamellae. 

Infection  in  the  field  appears  to  take  place  through  wounds  in  the 
skin,  and  especially  through  cracks  in  the  skin  and  flesh. 

CAUSAL  ORGANISM. — According  to  Giddings,  Bacillus  melonis  possesses  the 
following  characteristics: 

A  bacillus  I.O/A  to  i.y/i  by  o.6/*  to  o.gn,  actively  motile  by  4  to  6  peritrichate 
flagella.  Endospores  not  produced.  Gram-negative.  Stains  readily  with  aqueous 
stains. 

In  nutrient  broth,  strong  clouding  twenty-four  hours,  neither  pellicle  nor  ringk 
slight  sediment.  Agar  stroke,  abundant,  contoured,  shiny,  glistening,  without  color, 
opalescent  growth  having  umbilicate  elevation.  Gelatin  stab,  infundibuliform 
liquefaction  in  two  days.  Cooked  potato,  abundant,  spreading,  glistening,  odor  of 
decaying  potatoes.  Litmus  milk,  coagulated  and  reddened  in  three  days,  no  di- 
gestion. No  growth  in  Cohn's  solution.  Abundant  growth  in  Uschinsky's  solution, 
ring,  pellicle  and  heavy  sediment,  odor  of  hydrogen  sulphide.  Vegetables  rotted — 
muskmelon,  citron,  carrot,  potato,  beetf  and  turnip.  Growth  and  some  acid  but 
no  gas  from  lactose,  etc.  Slight  gas  production  from  asparagin  broth,  abundant  in 
fermentation  tubes  of  milk,  this  gas  being  99  per  cent  carbon  dioxide.  Hydrogen 
sulphide  from  nutrient  broth  and  potato.  Nitrates  reduced.  Slight  indol.  Am- 
monia from  asparagin  broth;  none  from  broth,  gelatin,  milk  or  urea.  Thermal 
death-point,  49°  to  50°.  Optimum  temperature,  30°. 

CONTROL. — Spraying  with  Bordeaux  mixture  or  other  fungicides  is 
recommended  as  a  preventive  measure. 

*  Giddings,  Bull.  148,  Vermont  Exp.  Station,  1910. 

t  B.  carotovorus.  Jones,  associated  with  several  soft  rots,  does  not  rot  the  beet. 


ROTS  987 

The  melons  should  be  supported  by  some  means  to  keep  them  from 
coming  in  direct  contact  with  the  soil,  and  should  be  supplied  with 
adequate  water  during  a  dry  season  to  keep  them  from  cracking. 

SOFT  ROT  OF  THE  SUGAR  BEET 
Bacterium  teutlium — Metcalf 

HISTORY. — A  soft  rot  of  the  sugar  beet,  occurring  in  Nebraska,  has 
been  described  by  Metcalf  and  Hedgcock.* 

SYMPTOMS. — Beets  affected  with  the  rot  show  the  lower  half  badly 
decayed  and  honeycombed  with  "pockets"  or  cavities  rilled  with  a 
slimy,  stringy  fluid,  colorless,  sour-smelling,  and  alive  with  bacteria. 
The  vascular  bundles  remain  intact,  while  the  tissue  surrounding  them 
is  usually  consumed.  Above  ground  the  beets  appear  normal. 

METHOD  OF  INFECTION. — The  germs  gain  entrance  to  the  beet 
through  wounds  and  abrasions  in  the  skin,  and  there  is  good  reason  for 
believing  that  nematodes  are  responsible  for  many  of  the  inoculations. 

CAUSAL  ORGANISM. — Bacterium  teutlium,  according  to  Metcalf,  possesses  the 
following  characteristics: 

It  is  a  short,  non-motile  rod,  rounded  ends,  1.5/1  by  o.8/*;  neither  capsules  nor 
endospores  have  been  observed;  the  organism  stains  readily  with  the  aqueous 
stains.  Gram-positive. 

On  nutrient  agar,  slow,  scant,  translucent,  porcelain  white,  non-viscid,  and  pene- 
trates the  agar.  On  cane-sugar  agar  growth  more  rapid,  viscid,  watery,  vitreous  to 
translucent,  colorless.  Gelatin  stab — scant,  filiform  to  beaded,  dirty  white,  no 
liquefaction.  Cane  sugar  gelatin — characteristic  cumulus  cloud  appearance  in 
stab,  no  liquefaction.  Nutrient  broth — slight  clouding  and  sediment,  acid  pro- 
duced. No  evidence  of  growth  in  milk.  No  visible  growth  on  potato.  On  carrot, 
clear,  viscid  and  acid.  On  sugar  beet,  viscid,  clear,  spreading,  copious,  acid, 
parenchyma  destroyed  leaving  vascular  tissue.  No  growth  in  Uschinsky's,  Fermi's, 
Pasteur's,  Fraenkel's,  or  Dunham's  solution.  No  gas  from  dextrose,  saccharose, 
etc.  Facultative  anaerobe.  No  growth  at  37°.  Optimum  temperature,  17°. 
Thermal  death-point,  45°. 

CONTROL. — -The  rot  is  less  apt  to  be  serious  if  the  beets  are  grown  on 
relatively  dry  soil  and  if  rotation  of  crops  is  practiced.  The  selection 
of  resistant  varieties  seems  to  be  the  most  practical  solution  of  the 
problem. 

*•  Metcalf  and  Hedgcock,  "A  Soft  Rot  of  the  Sugar  Beet,"  i?th  Annual  Report,  Nebraska 
Agr.  Exp.  Sta.,  pp.  69-112,  1904. 


CHAPTER  V 
WILTS 

WILT  or  CUCURBITS 
Bacillus  tracheiphilus — Erw.  Smith 

HISTORY  AND  DISTRIBUTION. — The  bacterial  wilt  of  the  muskmelon, 
cucumber,  squash  and  pumpkin  was  first  reported  by  Erwin  Smith*  in 
1893.  It  is  widely  distributed  over  the  United  States  east  of  the  Rocky 
Mountains  and  seems  to  have  different  host  preferences  in  different 
localities. 

SYMPTOMS. — The  disease  is  characterized  by  a  wilting  of  the  vine, 
pure  and  simple,  without  any  visible  external  cause  such  as  mildew, 
rust  or  leaf  spot.  The  leaves  and  runners  wilt  suddenly  as  if  from  lack 
of  water  or  too  hot  sun,  the  runner  becoming  prostrate  on  the  ground. 
From  two  to  three  days  usually  elapse  before  the  wilting  of  the  whole 
vine  is  complete,  and  it  may  remain  in  this  wilted  condition  for  several 
days,  after  which  the  leaves  begin  to  dry  up,  but  retain  their  green 
color  for  considerable  time.  One  runner  may  die  at  a  time,  beginning 
at  the  tip  and  working  back  toward  the  root,  after  which  a  general 
infection  is  to  be  expected.  If  inoculation  takes  place  upon  the  main 
stem,  several  or  all  of  the  runners  may  show  the  wilt  at  the  same, 
time. 

The  disease  is  caused  by  a  bacillus  whose  growth  fills  the  water  ducts 
or  tracheae  with  a  white,  viscid  material  which  prevents  the  rise  of 
water,  and  wilting  follows.  If  the  severed  ends  of  a  diseased  vine  are 
rubbed  together  gently  and  separated  slowly,  this  sticky  liquid  will 
string  out  in  fine  threads  2  to  3  cm.  in  length. 

METHOD  or  INFECTION. — Under  field  conditions,  the  disease  is 
spread  principally  by  insects,  especially  the  striped  cucumber  beetle 
and  the  common  squash  bug. 

*  Smith,  Erwin:  Cent.  f.  Bakt.,  Bd.  I,  II.,  Abt.,  pp.  364-373,  1895. 

988 


WILTS  989 

CAUSAL  ORGANISM. — Erwin  F.  Smith  describes  Bacillus  tracheiphilus  as  a  rod 
i.2ju  to  2.5/u  by  O.SJLI  to  o.7ju,  actively  motile  when  young. 

Growth  occurs  on  the  ordinary  media.  Upon  agar,  the  growth  is  milk-white  and 
extremely  viscid.  Upon  potato,  a  gray  film  is  produced,  much  like  that  of  B. 
typhosus;  the  potato  is  unchanged.  Gelatin  is  liquefied  and  no  change  occurs  in 
milk.  Acid  but  no  gas  is  produced  in  saccharose  and  dextrose  broths.  The  organ- 
ism is  aerobic  and  possibly  facultatively  anaerobic.  Optimum  temperature  is 
between  20°  and  30°.  No  growth  at  37°.  Thermal  death-point,  43°. 

CONTROL. — The  same  precautions  and  preventive  measures  are  to 
be  recommended  for  the  wilt  of  cucurbits  as  are  given  for  tomato 
blight. 

WILT  OF  SWEET  CORN 
Pseudomonas  stewarti — Smith 

The  early  varieties  of  sweet  corn  grown  in  the  truck  gardens  of  Long 
Island*  are  subject  to  a  bacterial  disease  which  manifests  itself  by  a 
wilting  and  drying  up  of  the  leaves.  It  also  occurs  in  Iowa,  and  it 
has  been  reported  from  certain  parts  of  New  Jersey. 

The  wilting  may  occur  at  any  stage  of  growth,  but  the  plants  seem 
to  be  more  susceptible  at  the  time  of  flowering.  As  a  rule  the  leaves 
succumb  one  at  a  time,  although  on  the  younger  plants  they  may  all 
wilt  simultaneously.  There  is  no  external  evidence  which  would 
indicate  the  cause  of  the  trouble,  but  if  a  diseased  stalk  is  cut  length- 
wise, the  fibro- vascular  bundles  appear  as  yellow  strands  in  the  white 
pith.  A  cross-section  of  such  a  stalk  will  show  drops  "of  a  yellow  viscid 
substance,  composed  largely  of  bacteria,  exuding  from  the  cut  ends  of  the 
bundles.  The  infection  is  not  confined  to  the  stalks  but  can  be  found 
in  the  vascular  system  of  the  leaves,  husks  and  cobs  as  well.  The 
vessels  are  the  principal  structures  invaded,  but  in  time  small  cavities 
filled  with  the  bright  yellow  slime  are  formed  in  the  surrounding 
parenchyma. 

METHOD  OF  INFECTION. — The  germ  may  enter  its  host  through 
either  the  roots,  stomata  or  water  pores  and  when  once  inside  the  vas- 
cular system,  it  multiplies  very  rapidly,  fills  the  water  tubes  with  a 
yellow  slime  and  wilting  follows. 

CAUSAL  ORGANISM. — The  organism  was  first  described  by  Stewart  and  later 
named  Pseudomonas  stewarti  by  Erwin  Smith. 

*  Stewart,  P.  C.,  "A  Bacterial  Disease  of  Sweet  Corn."  Bull.  130,  N.  Y.  Agr.  Exp.  Sta.,  1897. 


9QO  MICROBIAL  DISEASES    OF   PLANTS 

It  is  a  short,  relatively  thick,  motile  rod  with  rounded  ends;  occurs  usually  in 
pairs.  No  endospores  observed.  Stains  readily  with  the  aqueous  stains. 

It  grows  well  upon  the  ordinary  culture  media.  On  agar,  smooth,  shining, 
yellowish-white  to  deep  yellow,  lobate.  On  potato,  spreading,  deep  yellow  be- 
coming slightly  iridescent,  smooth;  potato  is  browned.  Broth — thin  film,  slight 
clouding  and  slight  flocculent  white  precipitate.  Milk — slight  peptonization  with- 
out coagulation;  litmus  reduced.  No  gas  is  produced  from  dextrose,  etc.  Good 
growth  in  Uschinsky's  solution.  Facultative  anaerobe.  Pathogenic  for  sweet 
corn. 

CONTROL. — It  is  believed  that  the  germ  is  disseminated  on  dis- 
eased seed  and  therefore  disinfection  of  the  seed  before  planting  is 
recommended. 

The  disease  is  also  spread  by  the  use  of  manure  which  contains 
diseased  stalks. 

Varieties  differ  considerably  in  their  susceptibility,  and  by  the 
selection  of  the  more  resistant  kinds  some  relief  can  be  secured. 

Rotation  of  crops  and  planting  on  new  land,  when  available,  should 
be  practiced. 

Field  corn  and  pop-corn  are  not  affected  by  the  wilt. 

WILT  OF  TOMATO,  EGGPLANT,  IRISH  POTATO  AND  TOBACCO 
Pseudomonas  solanacearum — Erwin  Smith 

HISTORY. — A  bacterial  wilt  affecting  a  number  of  plants  of  the 
potato  family  has  been  described  by  Erwin  Smith.*  The  disease  was 
first  observed  in  the  Atlantic  coast  and  southern  states.  In  1903 
Stevensf  and  Sackett  described  a  wilt  of  tobacco  in  Granville  County, 
N.  C.  and  this,  too,  SmithJ  has  shown  to  be  due  to  the  tomato  wilt 
organism,  Ps.  solanacearum.  Quite  recently  Miss  Bryan§  has  shown 
the  same  organism  to  be  the  cause  of  nasturtium  wilt. 

SYMPTOMS. — The  disease  usually  manifests  itself  by  a  sudden  wilting 
of  the  foliage,  and,  as  a  rule,  with  little  or  no  yellowing.  This  may  be 
indicated  at  first  by  the  collapse  of  a  single  leaf,  but  in  time  the  whole 
plant  will  succumb.  Following  the  wilting,  the  parts  affected  shrivel, 

*  Smith,  Erwin  P.,  "A  Bacterial  Disease  of  the  Tomato,  Eggplant  and  Irish  Potato,"  Bull. 
12,  U.  S.  Dept.  Agr.,  Div.  Veg.  Phys.  and  Path.,  1896. 

t  Stevens  and  Sackett,  "Granville  Tobacco  Wilt,"  Bull.  188,  N.  Car.  Exp.  Sta.,  1903. 

J  "Granville  Tobacco  Wilt,"  Bull.  141,  U.  S.  Dept.  Agr.,  Bur.  Plant  Industry,  1908." 

§  Bryan,  Mary  K.,  "A  Nasturtium  Wilt  Caused  by  Bact.  Solanacearum,"  Jour,  of  Agr. 
Research,  Vol.  IV,  No.  5,  p.  45L  IQIS 


WILTS  991 

turn  yellow,  then  brown,  and  finally  black.  If  a  diseased  stem  is  split 
lengthwise,  black  streaks,  following  the  fibre-vascular  bundles,  can  be 
traced  the  whole  length  of  the  stem  and  often  out  into  the  corresponding 
leaves.  The  vessels  are  packed  with  bacteria  which  ooze  out  on  the  cut 
surface  as  little  drops  of  a  dirty  white,  slightly  viscid  liquid.  The 
bacillus  destroys  the  parenchyma  of  the  pith  and  bark  and  mechanically 
plugs  the  water  tubes  so  that  the  water  supply  from  the  soil  is  shut  off 
and  wilting  follows.  In  the  tubers  of  the  potato,  the  rot  begins  in  the 
blackened  vascular  ring  and  spreads  in  all  directions,  producing  well- 
defined  cavities  next  to  the  ring. 

METHOD  OF  INFECTION. — Insect  enemies  are  largely  responsible  for 
the  spread  of  the  wilt,  especially  above  ground,  while  beneath  the 
.surface  inoculated  soil  enters  the  roots  through  wounds  made  either  by 
transplanting,  cultivating,  or  nematodes.  In  the  case  of  the  nastur- 
tium, stomatal  infections  have  been  demonstrated. 

CAUSAL  ORGANISM. — According  to  Smith,  Pseudomonas  solanacearum  is  a 
medium-sized  rod,  rounded  ends;  1.5/11  by  0.5/1;  motile  by  a  single  polar  flagellum, 
zooglceae  formed  in  liquid  media;  stains  readily  with  aqueous  stains. 

Zooglceae  produced  at  the  surface  in  beef  broth,  copious  dirty  white  sediment, 
reaction  made  alkaline.  Casein  of  milk  dissolved  without  precipitation  and  medium 
becomes  alkaline.  On  nutrient  agar,  growth  is  smooth,  wet  shining,  slightly  viscid, 
at  first  dirty  white  becoming  yellowish,  then  brown;  agar  browned.  Gelatin 
stab — growth  best  at  surface,  pure  white,  smooth,  wet  shining,  no  liquefaction  or 
very  feeble  after  six  weeks.  Potato — wet  shining,  not  wrinkled,  copious,  dirty 
white  and  later  brown  to  black;  medium  browned.  Neither  acid  nor  gas  produced 
in  any  of  the  culture  media  or  from  glucose,  etc.  Obligate  aerobe;  ammonia  pro- 
duced in  nutrient  broth  and  potato  tubes;  pigment  formation  aided  by  glucose, 
fructose  and  saccharose.  Grows  well  at  37°.  Thermal  death-point,  52°. 

PATHOGENESIS. — Pathogenic  for  tomato,  potato,  eggplant,  tobacco, 
Jamestown  weed,  black  nightshade,  physilis,  petunia  and  nasturtium. 

•CONTROL. — If  the  disease  is  not  too  general,  it  is  possible  to  control 
its  spread  by  removing  the  dead  plants  and  burning  them;  the  early  and 
complete  destruction  of  all  insect  pests  is  important;  if  available  and 
practical,  new  land  or  land  which  has  not  been  planted  to  any  of  the 
potato  family  for  a  period  of  years,  should  be  used;  only  those  seeds  and 
tubers  which  have  come  from  plants  grown  in  localities  free  from  the 
disease  should  be  planted;  the  use  of  infected  manure  or  soil  should  be 
avoided. 


9Q2  MICROBIAL  DISEASES  OF  PLANTS 

ADDITIONAL  BACTERIAL  DISEASES 

Angular  Leaf  Spot  of  Cotton,  Pseudomonas  mahacearum  Smith.* 

Gum  Disease  of  Sugar  Cane,  Pseudomonas  vascularum  Cobb,f  Smith.  % 

Leaf  Spot  of  Broom  Corn,  Burrill.  § 

Bacteriosis  of  Tomatoes,  Bacillus  briosii  Pavarino.|| 

Wilt  of  Banana  and  Plantins,  Bacillus  muses  Rorer.** 

Bacteriosis  of  Ixia  maculata,  Bacillus  ixia  Severini.ft 

Bacteriosis  of  Gladiolus  cohilli,  Pseudomonas  gladioli  Severini.ft 

Bacteriosis  of  Orchard  Grass,  Bacterium  rathayi  Smith. \\ 

Rot  of  Potatoes,  Bacillus  solanisaprus  Harrison.  §§ 

A  Bacterial  Disease  of  the  Mango,  B.  mangifera.     Doidge.|||| 

*  Smith,  Erw.,  Bacteria  in  Relation  to  Plant  Diseases,  I,  p.  95,  126. 

t  Cobb,  N.  A.,  Rept.  New  So.  Wales  Dept.  Agr.,  1893,  pp.  1-21. 

t  Smith,  Erw.,  Cent.  f.  Bakt.,  II  Abt.,  Bd.  XIII,  22-23,  pp.  726-729,  1904. 

§  Burrill,  Bull.  6,  111.  Exp.  Sta.,  pp.  .165-176,  1889.  Smith  and  Hedges,  Science,  N.  S., 
Vol.  XXI,  535,  p.  502,  1905. 

||  Pavarino,  G.  L.,  Atti  R.  Accad.  Lincei.  Rend.  Cl.  Sci.  Fis.,  Mat.  e  Nat.,  5,  ser.,  20  (1911), 
I,  No.  s,  PP.  3SS-3S8. 

**  Rorer,  J.  B.,  Phytopathology,  I,  (1911)1  No.  2,  pp.  45~49. 

ft  Severini,  G.,  Ann.  Bot.  (Rome),  n  (1913),  No.  3,  pp.  413-424. 

tt  Smith,  Erw.  P.,  "A  New  Type  of  Bacterial  Disease."  Science,  N.  S.,  Vol.  XXXVIII, 
No.  991,  p.  926,  1913;  Sitz.  Ber.  Weiner  Akad.,  i  Abt.,  Bd.  CVIII,  p.  597. 

§§  Harrison,  F.  C.,  "A  Bacterial  Rot  of  the  Potato  Caused  by  Bacillus  solanisaprus."  Cent, 
f.  Bakt.,  Abt.  II,  Bd.  17,  p.  34,  1907. 

||||Doidge,  Ethel  M.,  Annals  of  Applied  Biology,  Vol.  II,  No.  i.  May,  1915,  PP.  1-45. 


INDEX  OF  CONTRIBUTORS 


BIOLETTI,  FREDERIC  T.,  61,  579,  580, 

603,  658 

BUCHANAN,  R.  E.,  303,  516 
CRUESS,  W.  V.,    559,  564,  570,  572, 

574.  577,  578,  649,  651,  652, 

654,  655 
DORSET,  M.,  119,  832,  838,  856,  858, 

859,  860,  863,  864,   874,  875 
EDWARDS,  S.  F.,  412,  524 
FIDLAR,  EDWARD,  781,  785,  787,  791, 

792,  795,  798,  812,  816,  822, 

825,  828,  830,  839,  842,  845, 

851 
FROST,  W.  D.,  79 

GUILLIERMOND,  A.,   15,  40,  63,  90 

HARRISON,  F.  C.,  i,  310,  801,  807,  818 
HASTINGS,  E.  G.,  444,  474,  486 
HILL,  H.  W.,  754,  854 


ITANO,  ARAO,  145 
KING,  WALTER  E.,  724,  740,  857 
LIPMAN,  JACOB  G.,  345 
MACNEAL,  W.  J.t  542,  550,  581,  593, 
810,  865,  868 

MCCAMPBELL,  E.  F.,  659,  684 

MARSHALL,  C.  E.,  11,  99,  145,  195 
PHELPS,  EARLE  B.,  330 
RAHN,  OTTO,  195,  263,  286,  297 
RETTGER,  L.  F.,  806 
REYNOLDS,  M.  H.,  778,  782,  784,  789, 
809,  819,  825,  833,  839,  873 
SACKETT,  WALTER  G.,  949 
STOCKING,  W.  H.,  428,  504 
THOM,  CHARLES,    13,   36,   775,   776, 

777,  944 

4TODD,  J.  L.,  13,  123,  197,  876,  969 
WYANT,  Z..  NORTHRUP,  905 


*  During  Dr.  Todd's  absence  on  active  service,  Dr.  E.  E.  Tyzzer,  George  Fabyan  Professor 
of  Comparative  Pathology,  Harvard  University,  revised  the  sections  dealing  with  protozoa. 
The  contributors  wish  to  express  their  appreciative  thanks  to  Dr.  Tyzzer  for  his  generous  and 
able  cooperation. 


t>3 


993 


INDEX  OF  SUBJECTS 


ABNORMAL  fermentation  of  milk,  463 
Abortion,  contagious,  810 
Abscesses,  792 
Acanthocystis  aculeata,  28,  29 

karyokinesis  of,  29 
Acetic  acid  as  preservative,  556 
production  of,  649 

bacteria,  636 
in  wine,  609 

fermentation,  636 
Acetone,  production  of,  649 
Achorion  schonleinii,  in  favus,  778 
Achromatic  spindle,  28 
Acid-forming  bacteria  in  milk,  444 
Acid-gas  fermentation,  235 
Acidity,  160 

of  soils,  355 
Actinobacillosis,  781 
Actinomyces  albus,  in  soil,  363 

bo  vis,  in,  784 

in  actinomycosis,  778 
cultural  characteristics,  778 

chromogenus,  in  soil,  363,  364 

as  higher  bacteria,  1 1 1 

scabies,  limiting  reaction  of,  357 

in  soil,  363 
Actinomycosis,  778 

Activities  of  organisms,  products  of,  230 
Addition  of  nitrogen  to  soil,  374 
Adsorption,  171 
Ae'des  (stegomyia)  calopus,  876 
Aeration,  of  milk,  455 

of  soijls,  350 
Aerobic  organisms,  in  soil,  350 

in  wine,  608 
Agglutinate,  716 
Agglutination,  stages  of,  717 

substances  concerned  in,  715 

suspensions  for  test,  751 


Agglutination,  test  for  glanders,  752 

test  for  other  diseases,  752 

test  for  typhoid,  752,  846 
Agglutinins,  714,  751 

co-,  716 

distribution  in  blood,  715 

hemo-,  717 

inherited,  715 

in  leprosy,  829 

normal,  696,  714 

production  of,  714 

structure  of,  716 
Agglutinogen,  715 

structure  of,  716 
Agglutinoids,  717 
Aggressin,  blackleg,  730 
Agitation,  influence  of,  285 
Air,  carrier  of  contagion,  308,  664 

determination    of    microorganisms 
in,  305 

disease  organisms  in,  308 

entrance    of  microorganisms   into, 

3<>4 

fermentation  organisms  in,  309 
freeing  from  bacteria,  309 
kinds    of   microorganisms  in,   303, 

307,  308 

of  milk  house,  439 
number  of  microorganisms  in,  306 
occurrence    of   microorganisms   in, 

304 
subsidence    of   microorganisms   in, 

305 

Air-borne  infection,  664 
Alcohol,  distilled,  631 

enzymes  of,  208,  214 

fermentation  of,  634 

as  food,  224 

methods  of  preparation  of,  632 


995 


996 


INDEX 


Alcohol,  microorganisms  of,  635 

in  milk,  466 

as  preservative,  557 

saccharine  raw  materials  of,  632 

sources  of,  631 

starchy  raw  materials  of,  633 

uses  of,  631 
Alcoholase,  208,  214 
Alcoholic  fermentation,  234 

equation  of,  225 
Ales,  622 

Aleuria  cerea,  ascus  development,  44 
Alexins,  705 

Alfalfa,  stem  blight  of,  951 
Algae  in  soil,  364 
Alkalinity,  161 
Allorhina  nitida,  907 
Alternaria,  general  characters,  57 

tenuis,  362 

Aluminum  in  soil,  425 
Amboceptor,  706 

anti-,  707 

American  foul  brood,  920 
Amidase,  208,  215 
Amino-acetic  acid,  241 
Amino-acids,  as  food,  223 

production  of,  241 
Amins,  production  of,  241 
Amitosis,  28,  34 

in  yeasts,  66 
Ammonia,  as  food,  222,  223 

oxidation,  201 

production  of,  241 

transformation,  398 
Ammonincation,  383 

climatic  conditions,  influence  of,  384 

efficiency,  388 

efficiency  of  species,  387 

mechanism  of,  384 

by  molds  in  soil,  361 

numbers  involved  in,  385 

rate  of,  387 

soil  conditions,  influence  of,  384 

species  involved  in,  385 
Amceba  (See  also  entamceba.) 

buccalis,  594 


Amceba,  coll,  66 1 

in  faeces,  599,  601 

diplomitotica,  protomitosis  of,  31 

dysenteriae,  in  intestine,  600,  601 

froschi,  protomitosis  of,  31 

histolytica,  600 

limax,  cyst  of,  24 

mucicola,  mitosis  of,  32 
protomitosis  of,  31 

polypodia,  128 
schizogony  in,  27 

proteus,  197 

terricola,  365 

tetragena,  66 1 

vespertilio,  124 
Amoebic  dysentery,  876 
Amylase,  208,  237 
Amylobacter,  376 
Amylomyces  rouxii,  50 
Amyloplastids,  20 
Amylo  process,  633 
Anabolism,  200,  203 

enzymic  theory  of,  217 
Anaerobic  organisms,  facultative,  228 

obligate,  228 

in  soil,  350 

in  wine,  609 
Anaerobic  tank,  342 
Analysis  of  milk,  470 

of  water,  321 
Anaphase,  28 
Anaphylaxis,  684 
Anaplasma  marginale,  898 
Anaplasmosis,  898 
Ang-quac,  56 
Anguillula  aceti,  647 
Anion,  159,  160 
Anisogamous  fertilization,  131 
Anode,  160 
Anopheles,  893 
Antagonism,  426 
Antheridium,  39 
Anthrax,  801-806 

symptomatic,  839-841 

vaccination,  805 

vaccine,  735 


INDEX 


.997 


Antiamboceptor,  707 
Antibacterial  substances,  natural,  695 
Antibiosis,  299 
Antibodies,  anti-,  707,  719 

occurrence  of,  698 

origin  of,  698 

test  for,  708 

Antibody  formation,  683 
Anticomplements,  708 
Anticytotoxin,  710 
Antidiphtheritic  serum,  741 
Antidysenteric  serum,  747 
Anti-gas  gangrene  serum,  745 
Antigens,  698 

for  diagnostic  tests,  753 
Antigonococcic  serum,  746 
Anti-hog  cholera  serum,  746 
Antimeningococcic  serum,  745 
Antimicrobial  serum,  740 
Antiperfringens  serum,  745 
Antipneumococcic  serum,  746 
Antiprecipitins,  719 
Antirabic  serum,  746 
Antiseptics,  288 
Antiserum,  740 
Antistreptococcic  serum,  745 
Antitetanic  serum,  741 
Antitoxin,  699,  740 

concentration  of,  744 

for  diphtheria,  741 

for  gas  gangrene,  745 

natural,  694 

neutralization  of  toxin  by,  702 

perfringens,  745 

preservation  of,  747 

for  tetanus,  744 

ynits  of,  703,  743,  -744 
Archoplasm,  16 
Arctia  caja,  918 
Arenicola  ecaudata,  929 
Argas  persicus,  901,  904 
Aromatic  substances,  248 
Arrack,  74,  632 
Arsin,  245 
Ascomycetes,  61 

cell  structure,  41 


Ascomycetes,  metachromatic  corpuscles 

in,  45 

reproduction  of,  39 
Ascospores,  germination  of,  67 

sporulation  of,  67 
Ascus,  39 

Ash  elements  in  cells,  192 
Asiatic  cholera,  851 

vaccine,  738 
Asparagin,  as  food,  223 
Aspergillosis,  775 
Aspergillus,  61 
candidus,  56 
characters,  54 
effect  of  light  on,  280 
flavus,  56 

in  aspergillosis,  775 
fruiting  bodies,  39 
fumigatus,  48,  55 

in  aspergillosis,  775 
glaucus,  55 
herbariorum,  55 
nidulans,  55 

in  aspergillosis,  775 
niger,  54,  374,  379,  387 
in  aspergillosis,  775 
chemotropism  in,  286 
conidial  organ  of,  45 
enzymes  of,  213,  215,  220 
metachromatic  corpuscles  of,  45 
nitrogen  fixation  by,  362 
oxidation  of,  234 
temperatures,  270 
.  ochraceus,  56 

energids  in,  16 
oryzae,  56 

proteolytic  enzyme  of,  213 
in  rice  beer,  630 
repens,  55 
terreus,  56 
wentii,  56 
Aster,  28 
Atta  sexdens,  918 
Autocytotoxin,  710 
Autogamy,  131 
Autohemoopsonins.  714 


998, 


INDEX 


Autolysis,  215 

Autolytic  enzymes,  208,  516 

Autoprecipitins,  719 

Autotrophic"  organisms,  223 

Avenue  of  infection,  674 

Avian  coccidiosis,  889 

Azotobacter,    371,  372,  374,  378,^380, 

398,  402,  403,  405.425 
*.-,      agilis,  403 

beyerincki,  404,  405 
^    .'..  chroococcum,  403,  404,  405,  420 
complete  oxidation,  234 
isolation  of,  359 
&  life  cycle  of,  100 
,     limiting  reaction,  357 
prototrophic,  223 
vinelandii,  403,  405 
vitreum,  404 
woodstownii,  404 

BABESIA,  139 

bigemina,  morphology,  895 

in  red  water,  894 
parva,  in  East  coast  fever,  896 
pathogenic,  894 
Bacillary     septicaemia    of    caterpillars, 

918 

white  diarrhoea,  806 
Bacillus  acetigenus,  638 

acridiorum,  905,  918,  946 

artificial  infection  with,  914-917 

in  disease  of  locusts,  912 

morphology  of,  913 
alvei,  96,  948 

in  European  foul  brood,  928 

metachromatic  corpuscles  of,  101, 

102 

amethystinus,  312 
amylobacter,  402,  404 

fermentation  of,  236 

fermentation  products  of,  233 

prototrophic,  223 

in  retting,  658 

in  sewage,  334 
amylovorus,  958 

morphology  of,  961 


Bacillus  amylozyma,  232 

anthracis  symptomatici  (B.  chau- 
vaei),  740 

in  blackleg,  839 

morphology  of,  841 

production  of  vaccine,  729 

toxin  of,  840 
aquatilis,  312 
aroideae,  984 
asterosporus,  96 

metachromatic  corpuscles  of,  ioi, 

102 

aurantiacus,  312 
bifidus,  in  faeces,  598 

in  intestine,  597 

pathogenic  in  intestine,  600 

technique  of  isolation,  600 
botulinus,  577,  663,  740 

acid  formation  of,  236 

antitoxin  for,  704 

in  botulism,  587 

on  cabbage,  571 

in  canned  foods,  529 

cultural  characteristics,  589 

in  food  poisoning,  584,  585 

incidence  of,  590 

indication  of  presence,  540 

morphology,  589 

in  olives,  573 

prevalence,  529 

in  silage,  576 

spoilage  due  to,  591 

in  stomach,  671 

toxin  of,  590,  676 

bronchisepticus,  production  of  vac- 
cine, 738 
biitschlii,  95  • 

diffuse  nucleus  in,  94 

membrane,  103 

nucleus  in,  97 
butyricus,  in  sewage,  334 
cacosmus,  in  fowl  diphtheria,  818 
cajae,  morphology  of,  918-919 

in  septicaemia  of  caterpillar,  918 
capsulatus  mucosus,  in  pneumonia, 
798 


INDEX 


999 


Bacillus  carotovorus,  984 

morphology,  985 
caucasicus,  morphology  of,  510 
chancroidae  mollis,  692 
chauvaei     (B.    anthracis    sympto- 
matic!), 841 
cholerae  suis,  386 

morphology  of,  863 
circulans,  in  water,  311 
cloacae,  312 
cceruleus,  312 
coli,  272,  386 

agglutinins  in  blood,  687,  714 

in  bread,  569 

in  bud-rot  of  cocoanut,  981 

in  butter,  476,  482 

in  concentrated  milk,  506 

effect  of  light  on,  278 

effect  of  phenol  on,  293 

in  fasces,  598 

fermentation  of,  235 

fermentation  products  of,  233 

in  food  poisoning,  584,  585 

in  genital  tract,  672 

glycogen  in,  190 

inhibition  of  growth,  301 

in  intestine,  596,  597 

isolation  from  faeces,  600 

in  milk,  472,  473 

in  milk  poisoning,  587 

nitrogen  content,  190 

in  olives,  573 

oxidation  of,  226 

pathogenic  in  intestine,  600 

plasmolysis  in,  264 

in  pneumonia,  798 

in  protein  degradation,  243 

in  sewage,  330,  340 

symbiosis  of,  298 

test  of  water,  322 

in  water,  313 

weight,  222 
coli  serogenes,  in  butter,  478 

in  cheese,  489,  490 

morphology,  448 
coli  communis,  313 


Bacillus,  coli  communis,  in  milk,  445, 

463 

vaccine,  737 

coli  communis  verus,  313 
communior,  313 
cyanogenes,  pigment  of,  246 
cypripedii,  982 

morphology  of,  982-983 
danicus,  403 

delbruckii,    in    compressed    yeast, 
560,  562 

in  lactic  acid,  651 
delphini,  975 

morphology  of,  975 
denitrificans,  395 
diphtheriae   columbarium,   in   fowl 

diphtheria,  819 
diphtheriae     gallinarum,     in     fowl 

diphtheria,  819 
enteritidis,  agglutination  of,  716 

in  canned  foods,  529 

in  food  poisoning,  584,  585 

in  intestine,  600 

in  sewage,  330 

enteritidis  sporogenes,  test  of  water, 
322 

in  water,  313 
erausquinii,  946 
erodiens,  in  leather,  657 
flexilis,  sporulation  of,  98 
fluorescens,  239,  378 

plasmolysis  in,  264 

in  protein  degradation,  243 
fluorescens  liquefaciens,  387 

in  leather,  655 

in  water,  311 
fluorescens      non-liquefaciens,      in 

water,  311 

fluorescens  putidus,  387 
fulvus,  312 

furfuris,  in  leather,  657 
fusiformis,  in  mouth,  594,  595 
gangraenae     emphysematosae,     pro- 
duction of  vaccine,  729 
gortynae,  946 
graphitosis,  920 


1000 


INDEX 


Bacillus  hoplosternus,  945 
hyacinth!  septicus,  985 
indicus,  312 
indigogenus,  658 
industrius,  in  vinegar,  638 
invertenti  acetici,  in  acetone  pro- 
duction, 650 

invertenti  lattici,  in  acetone  pro- 
duction, 650 
janthinus,  312,  385,  387 

pigment  of,  246 
lactis  viscosus,  403,  465 

in  milk,  441,  464,  465 
larvae,  in  American  foul  brood,  921 

morphology,  923-924 
lathyri,  962 

morphology,  962 
leptosporus,  86 
lineola,  105 
liparis,  945 

liquefaciens,  in  water,  311 
lividus,  312 
lymantriae,  930,  945 
lymantricola  adiposus,  930 

morphplogy,  930 
macerans,   in   acetone  production, 

650 

malabarensis,  403 
maximus  (buccalis),  in  mouth,  594 
megatherium,  86,  96,  312,  386,  948 

in  leather,  655 

metachromatic  corpuscles  of,  101 

nucleus  of,  92 
melolonthae,  911,  925,  945 

morphology,  926 
melolonthae  liquefaciens,  945 
melolonthae  non-liquefaciens,  945 
melonis,  986 

morphology,  986 
mesentericus,  385,  403 

in  canned  food,  528 

in  soil,  370 

mesentericus  fuscus,  312 
mesentericus  ruber,  312 
mesentericus  vulgatus,  312,  387 

in  compressed  yeast,  562 


Bacillus  mesentericus  vulgatus,  in  ropy 

bread,  569 
methanicus,  377 
methylicus,  380 
mirabilis,  312 
mycoides,  17,  96,  312,  383,  420,  424 

in  air,  307 

ammonification  of,  385,  386,  387, 
388 

metachromatic  corpuscles  of,  101 

nucleus  of,  92 

in  protein  degradation,  242 

proteolytic  enzyme  of,  213 

in  soil,  370 

spore  formation  in,  27 
nitrator,  391 
non-liquefaciens,  945 
ochraceus,  312 
cedematis  maligni,  in  faeces,   598, 

599 

in  intestine,  597 

morphology  of,  839 
oleraceae,  983 

morphology,  983 
oligocarbophilus,  378 
omelianski,  in  sewage,  334 
orpheus,  in  European  foul  brood, 

928 

oxydans,  in  vinegar,  638 
paracoli,  agglutination  of,  716 
paralacticus,  in  yahourth,  511 
paratyphosus,  agglutination  of,  716 

in  fish  poisoning,  586 

in  food  poisoning,  584,  585 

in  intestine,  600 

in  milk  poisoning,  587 

production  of  vaccine,  737 
Bacillus  (clostridium)  pasteurianus,  402, 
404 

in  soil,  378 
phytophthorus,  980 

morphology,  981 
pieris  agilis,  945 
pieris  fluorescens,  945 
pieris  liquefaciens,  945 
pieris  non-liquefaciens,  945 


INDEX 


1001 


Bacillus  pluton,  in  European  foul  brood, 
926 

morphology,  928 
poncei,  947 
prodigiosus,  80,  239,  403 

in  air,  305 

ash  elements  in,  193 

on  bread,  569 

effect  of  light  on,  278 

oxygen  pressure,  284 

pigment  of,  246 

in  protein  degradation,  243 

proteolytic  enzyme  of,  213 

in  sewage,  340 

in  water,  311 
proteus,  in  protein    decomposition, 

243 

proteus  mirabilis,  in  leather,  655 
punctatus,  in  water,  311 
putrificus,  in  protein  degradation, 

243 

pyrameis,  946 

radicicola  (See  pseudomonas.) 
radicosus,  96,  102 

metachromatic  corpuscles  in,  101, 
102 

nucleus  in,  92 
radiobacter,  403 
ruber,  in  water,  311 
rubescens,  312 
rubifaciens,  312 
rudensis,  in  cheese,  499 
ruminatus,  403 
saccobrinchi,  98 
septicus  insectorum,  929 

morphology,  929 
simplex,  403 
sotto,  945,  948 
spirogyra,  99 
sporonema,  95,  96 
subtilis,  86,  312,  385,  386,  387 

in  air,  307 

in  compressed  yeast,  561,  562 

in  disease  of  locusts,  913 

effect  of  gravity  on,  284 

in  faeces,  598 


Bacillus  subtilis,  glycogen  in,  190 

in  leather,  655,  657 

organs  of  locomotion,  25 

plasmolysis  of,  264 

in  soil,  354,  370 

temperatures  for,  270 
suipestifer,  morphology,  863 
sulphureus,  in  sewage,  336 
tetani,  663,  726 

acid  formation  of,  236 

antitoxin  for,  704 

for  antitoxin  production,  744 

facultative  parasite,  660 

morphology,  843 

on  skin,  689 

test  of  vaccine  for,  728 

in  tetanus,  843 

toxin  of,  676 
tentlium,  987 

morphology,  987 
tracheiphilus,  morphology  of,  989 

in  wilt  of  cucurbits,  988 
tracheitis,  in  graphitosis,  920 

morphology  of,  920-921 
tumescens,  388 

fat  content,  192 

in  soil,  370 
typhosus,  662,  687,  705,  726 

agglutination  of,  752,  846 

agglutination  test  for,  716 

agglutinins  in  blood,   697,   714, 

715,  7i7 

effect  of  pressure  on,  284 
elimination  of,  697 
facultative  saprophyte,  660 
fermentation  of,  236 
flagella,  105 
in  food,  582 
.  in  food  infection,  666 
on  house  flies.  667 
human  carriers  of,  668 
in  intestine,  600,  671,  692 
isolation  from  faeces,  60 1 
morphology,  847 
in  pneumonia,  798 
precipitins  for,  718 


1002 


INDEX 


Bacillus  typhosus,  production  of  vac- 
cine, 737 

resistance  to,  675 

in  sewage,  340 

toxin  of,  676 

in  typhoid  fever,  845 

variation  in  infection,  673 

in  water,  314 
violaceus,  312 

pigment  of,  246 

proteolytic  enzyme  of,  213 
vulgaris,  312 

(Proteus)  vulgaris,  385,  386,  387, 
419,  424 

in  faeces,  598 

in  food  poisoning,  584,  585 

in  leather,  655     ' 

nitrogen  content,  189 

pathogenic  in  intestine,  600 
xerosis,  ash  elements  in,  193 
zeukeri,  312 
zopfi,  312 
Bacteria,  38,  79 

artificial  cultivation  of,  118 
ash  elements  in,  193 
cell  aggregates,  87,  88 

capsule,  103,  104 

cytoplasm,  89,  90 

flagella,  82,  105 

nucleus,  89,  90 

volume,  221 

wall,  102 

weight,  221 
chromogenic,  246 
classification  of,  in 
cytology  of,  89 
diseases  caused  by,  784 
on  grapes,  608 
higher,  107 

forms  of,  107 
involution  forms  of,  80 
life  cycle  of,  99 
lower,  79 

form  types  of,  79 

gradations,  80 

involution  forms,  80,  81 


Bacteria,  lower,  motion,  81 
brownian,  82 
character  of,  82 
organs  of,  82 
rate  of,  82 
vital,  82 
reproduction,  83 
spore  formation,  84 
vegetative,  83 
size,  81 

metachromatic  corpuscles,  101 
metachromatic  granules,  90 
nomenclature,  117 
pathogenic,  660 
relationship,  117 
reserve  products,  101 
in  soil,  367 

distribution,  368 
methods  of  counting,  370 
morphological  groups,  369 
numbers,  367 
physiological  groups,  370 
quantitative  relations,  391 
Bacterial  disease,  784 

of  gut-epithelium  of  lug- worm, 

929 

of  June  beetle  larvae,  906 
of  larvae  of  Lamellicornse,  929 
of  locusts,  912 
enteritis,  chronic,  809 
oxidation,  337 
substance,  in  soil,  397 

availability  in  soil,  398 
vaccines,  736 

Bactericidal  substances,  288,  704 
Bacterins,  736 
Bacteriolysins,  705 
Bacteriopurpurin,  247 
Bacteriosis  of  beans,  953 
Bacterium  abortus,  morphology  of,  810 
aceti,  morphology  of,  637 

in  white  lead,  654 
aerogenes,  272 
in  butter,  476 
fermentation,  235 
in  soil,  370 


INDEX 


IOO3 


Bacterium  anthracis,  86,  705,  707,  726 

in  anthrax,  801 

effect  of  disinfectant  on,  289 

effect  of  pressure  on,  283 

in  intestine,  600 

morphology,  802,  803 

nucleus  in,  92 

plasmolysis  in,  264 

production  of  vaccine,  735 

proteolytic  enzyme  of,  213 

temperatures,  270 

threads  of,  88 
arenicolae.  929 

morphology,  930 

bovisepticum,  in  haemorrhagic  sep- 
ticaemia, 825 

morphology  of,  827 
bulgaricum,  272 

in  artificial  buttermilk,  512 

in  cheese,  493,  496,  501,  502 

in  faeces,  599 

in  lactic  acid,  651,  652 

in  milk,  445 

morphology,  450 

in  yahourth,  511 

butyricum,  oxygen  pressure  on,  284 
capsulatus,  capsule  of,  104 
chlorinum,  118 

choleras    gallinarum,     in     chicken 
cholera,  807 

morphology,  808 
coli  apium,  946  . 
diphtheriae,  662,  726 

antitoxin  for,  704 

cell  aggregate,  88 

in  diphtheria,  812 

elimination  of,  679 

in  healthy  persons,  663 

metachromatic     corpuscles      of, 
101,  102 

morphology  of,  813 

in  mouth,  596 

in  nasal  cavity,  690 

in  pneumonia,  798 

precipitins  for,  718 

toxin  of,  676 


Bacterium    dysenteriae,    agglutinins    in 
blood,  697,  714 

cultural  characteristics,  817 

in  dysentery,  816 

in  intestine,  600 

in  milk,  470 

toxin  of,  676 
eurydice,  in  European  foul  brood, 

928 
gammari,  91 

nucleus  in,  91,  92 
influenzae,  in  conjunctiva,  692 

dissemination  of,  677 

in  healthy  persons,  663 

in  influenza,  822 

morphology,  824 

in  nasal  cavity,  690 

in  pneumonia,  798 

in  production  of  influenza-pneu- 
monia vaccine,  738 
kutznigianum,    in    vinegar,    mor- 
phology, 637,  638  -• 
lactis  acidi,  235,  273 

antibiosis  of,  300 

in  artificial  buttermilk,  512 

in  butter,  476,  477,  479,  483 

in  cheese,  489,  491,  496 

cultural  characteristics,  446 

in  ice  cream,  515 

in  kumyss-,,508 

in  milk,  432,  436,  445,  450,  462, 
463,  468 

in  yahourth,  511   • 
lactis  aerogenes,  in  milk,  445,  462, 

463 

in  water,  314 
leprae,  morphology,  828 

obligate  parasite,  660 
ludwigii,  temperatures,  270 
mallei,  in  glanders,  819 

in  mallein  production,  750 

morphology,  821 

in  nasal  cavity,  690 

toxic  protein  of,  676 

toxins  of,  820 
michiganense,  963 


INDEX 


Bacterium  murisepticum,  832 

necrophorus,  in  foot  rot  of  sheep, 

838 

panatuberculosis,  morphology,  809 
pasteurianum,    in    vinegar,    mor- 
phology, 637,  638 
pertussis,  in  whooping  cough,  825 

vaccine  for,  737 
pestis,  726 

avenue  of  entrance,  669,  675 

in  eye,  670 

in  fleas,  667 

in  intestine,  600 

morphology,  830 

precipitins  for,  718 

variation  in  infection,  673 
phosphoreum,    temperatures,    2  70, 

271 

pilline,  in  leather,  655 
pneumoniae,  403,  717 

capsule,  104 

moisture  content,  187 

variation  in  infection,  673 
pseudodiphtheriae,  707 

effect  of  pressure  on,  283 
pullorum,  806 

rhusiopathiae   suis,    in   swine    ery- 
sipelas, 832 
savastanoi,  969 
tuberculosis,  753 

ash  elements  in,  193 

carbohydrates  in,  190 

differentiation  of,  828 

droplet  infection  by,  665 

in  dust,  665 

effect  of  phenol  on,  293 

elimination  of,  678,  679 

fat  content  of,  192 

frequency  of  infection,  694 

in  healthy  persons,  663,  664 

in  intestine,  600,  671 

isolation  from  faeces,  601 

metachromatic     corpuscles      of, 
101,  102 

in  milk,  452,  470 

morphology,  836 


Bacterium  tuberculosis,  natural  suscep- 
tibility to,  685 
nitrogen  content,  189 
in  pneumonia,  798 
production  of  vaccine,  736 
toxic  proteins  of,  676 
in  tuberculin  production,  747 
in  tuberculosis,  833 
variation  in  infection,  672 
ureae,  in  sewage,  335 
vermiforme,  74 

in  ginger  beer,  631 
viride,  n8 
Welchii,  663,  740 

for  antitoxin  production,  745 
capsule,  104 
facultative  parasite,  660 
in  faeces,  598,  600 
in  intestine,  597,  600 
xylinum,  in  vinegar,  638 
Balantidium  coli,  morphology,  899 

enteritis,  899 
Barber's  itch,  777 
Bartonella  bacilliformis,  897 
Basidiomycetes,  reproduction  of,  39 
Basidium,  39 
Basophile  grains,  65 
Beans,  bacteriosis  of,  953 
Beef,  dried,  523 
extract,  523 

Beer,  after  treatment,  626 
ales,  622 
bacteria  of,  624 
brewing  of,  623 
composition  of,  622 
definition  of,  622 
diseases  of,  627 
enzymes  of,  623 
fermentation  of,  625 
grains  employed,  622 
lager,  622 
malting,  623 
pasteurization  of,  530 
porter,  622 
"temperance,"  622 
weisbiers,  622 


INDEX 


1005 


Beer,  wort,  623 

yeasts  of,  74,  622 
Bees,  nosema-disease  of,  939 
Beggiatoa  alba,  93 
Benzole  acid  as  preservative,  556 
Beriberi,  591 

Binary  fission  in  protozoa,  128 
Binuclearity  of  cells,  18 
Binucleata,  134 
Biological  activities,  physical  forces  in, 

155 

unit  of,  147 

Biological  changes  in  canned  foods,  528 
Bitter  cheese,  499 

milk,  465 
Blackleg,  839 

aggressin,  730 

filtrate,  731 

of  potato,  980 

vaccine,  729 
Blepharoplast,  25 
Blight  of  alfalfa,  951 

of  beans,  953 

of  lettuce,  954 

of  mulberry,  955 

of  oats,  956 

of  pears,  958 

(stem)  of  peas,  956 

(streak)  of  sweet  peas,  962 

of  tomato,  963 

of  walnut,  963 

Blood-forming  organs,  action  of  organ- 
isms on,  681 

Boas-Oppler  bacillus,  596 
Boda  ovatus,  365 
Boils,  792 

Bombyx  mori,  909,  910,  918,  937 
Boophilus  annulatus,  894 
Boric  acid  as  preservative,  555 
Botryomyces  equi,  784 
Botryomycosis,  784 
Botrytis  cinerea,  616 

in  wine,  604,  605 
Botulism,  587 
Brandy,  631 
Bread,  564 


Bread,  bacteria  of,  564 

diseases  of,  569 

dough,  565 

fermentation,  564 

French,  568 

Mexican,  568 

salt-rising,  569 

sour  dough,  568 

sponge  dough,  567 
Brewing,  623 
Bronchopneumonia,  795 
Brownian  motion,  172 
Bubonic  plague,  830 

vaccine,  739 
Budding,  27   ' 
Butter,  canned,  507 

chemical  preservation  of,  553 

decomposition  of,  483 

flavor  of,  475,  482 

-milk,  512 

pathogenic  bacteria  in,  485 

refrigeration,  547 

sour-cream,  474 

sweet-cream,  474 

types  of,  474 
Buttermilk,  artificial,  512 
Butyric  fermentation,  236 

equation  of,  232 

in  wine,  612 

CABBAGE,  black  rot  of,  978 

"stump  root"  of,  969 
Cadaverin,  241 
Calcium  in  soil,  417 
Calla  lily,  soft  rot  of,  984 
Calothrix  pulvinata,  nuclear  division  of, 

17 

Camembert  cheese,  502 
Canine  distemper,  856 

vaccine  for,  738 
Canker,  citrus,  973 
Canned  foods,  B.  botulinus  in,  529 

bacteria  in,  528 

biological  changes  in,  528 

butter,  507 

cheese,  507 


ioo6 


INDEX 


Canned  foods,  chemical  changes  in,  527 

corn,  534 

digestibility  of,  528 

fish,  534 

fruits,  535 

meat,  533 

olives,  573 

palatability,  528 

peas,  534 

physical  changes  in,  526 

processing  of,  532 

spoilage  of,  540 

sterilization  of,  532 

vegetables,  534 
Canning,  cleanliness  in,  535 

condition  of  raw  material,  535 

disposal  of  factory  refuse,  540 

heat  required  for,  536 

home,  539 

number    of     spores,     relation     of, 
536 

receptacles  for,  536 

resistance  of  spores,  536 

water  supply,  536 

Canning   of  foods,   commercial  impor- 
tance, 526 

dietetic  importance,  525 

economic  importance,  525 

health  importance,  525 

pasteurization,  530 
Capsules  of  bacteria,  104 
Carbohydrate  fermentation,  products  of, 
238 

foods,  drying  of,  521 
Carbohydrates,  in  cells,  190 

enzymes  of,  208,  209 

as  food,  223,  226 

in  soil,  375 
Carbon  cycle,  259 

dioxide,  as  food,  222,  223,  226 
oxidation  in  soil,  351 

monoxide,  as  food,  222,  224 

sources  for  organisms,  222 
Carbon-nitrogen  ratio  in  soils,  382 
Carchesium  polypinum,  19 
Carotin  bodies,  247 


Carriers  of  infection,  air,  664 

animal,  667 

food,  666 

human,  668 

milk,  467,  666 

water,  666 

Carrot,  soft  rot  of,  984 
Caterpillars,    bacillary    septicaemia    of, 

918 

Cathode,  160 
Cations,  159,  160 
Cattle  plague,  856 
Cauliflower,  rot  of,  983 
Causes  of  infection,  675 
Cell,  binuclearity  of,  18 

chemical  content,  186 

diffusion,  184 

ionization  in,  185 

mechanism,  151 

membrane,  103 

nutrition,  149 

osmosis  in,  177 

plasmolysis  in,  177 

reproduction,  27 

structure  of,  15 

as  unit  of  life,  147 

wall,  of  bacteria,  102 

of  molds,  47 
Cellulase,  208,  209 
Cellulose,  decomposition  by  molds,  362 

fermentation  of,  237 

in  microorganisms,  191 

in  soil,  375 

Centrifugal  separation  of  milk,  456 
Centriole,  16 
Centriole-nucleolus,  1 7 
Centrodesmose,  25 
Centrosome,  16 
Cercomonas,  135 

Cerebrospinal  meningitis,  epidemic,  787 
Certified  milk,  432 
Channels  of  infection,  659 
Character  of  milk,  428 
Cheddar  cheese,  500 
Cheese,  abnormal,  498 

acid-curd,  486 


INDEX 


I00y 


Cheese,  bitter,  499 

Camembert,  502 

canned,  507 

cause  of  proteolysis  of,  494 

Cheddar,  500 

colored,  499 

curdling  of  milk  for,  491 

Emmenthaler,  500 

flavor  of,  496 

gassy,  498 

Gorgonzola,  502 

kinds  of,  500 

moldy,  499 

poisoning,  586 

proteolysis  of,  494 

putrefaction  of,  495 

putrid,  499 

quality  of  milk  for,  487 

rennet-curd,  486 

ripening  of,  492 

ripening  of  milk  for,  490 

Roquefort,  502 

Stilton,  502 

Swiss,  500 

theories  of  ripening,  492 

types  of,  486 
Chelonia  caja,  945 

Chemical  changes  in  canned  foods,  527 
Chemical  content  of  cells,  186 

ash,  192 

carbohydrates,  190 

enzymes,  194 

fats,  191 

moisture,  187 

proteins,  188 

toxins,  194 

vitamines,  194 

Chemical  nature  of  food  poisons,  592 
Chemical  preservation  of  food,  550 

after  storage  changes,  551 

of  butter,  553 

effect  on  food,  550,  554 

of  fish,  551 

of  fruits,  553 

of  meat,  551 

storage,  551 


Chemical  preservation  of  vegetables,  553 
Chemicals,  influence  on  organisms,*286, 

326,  343 

Chemotaxis,  286 
Chemotropism,  286 
Chicken  cholera,  807 

pox,  854 

Chlamydobacteriaceae,  108 
Chlamydospores,  37 
Chlamydothrix  ochracea,  no 
Chlamydozoa,  139 

pathogenic,  900 
Chlorella  vulgaris,  moisture  content,  187 

nitrogen  content,  189 
Chloroplastids,  20 
Chloroplasts,  in  barley,  20 
Cholera,  Asiatic,  851 

chicken,  807 

hog,  860 

Chondriosomes,  19 
Chondrium,  19 

in  molds,  42 
Chromatic  reduction,  35 

in  protozoa,  130 
Chromatin,  16 

grains,  16 

in  protozoa,  125 
Chromatium  okenii,  93,  105 

cilia,  107 
Chromidium,  18 

in  protozoa,  18,  126 
Chromogens,  246 
Chromoparous  bacteria,  246 
Chromophorous  bacteria,  246 
Chromoplastids,  20 
Chromosomes,  28 

in  protozoa,  130 
Chronic  bacterial  enteritis,  809 
Chymosin,  208 
Cider,  alterations  in,  629 

composition  of,  628 

fermentation  of,  629 

microorganisms  of,  628 
Cilia,  25 

of  protozoa,  127 
Cirphis  unipuncta,  910 


ioo8 


INDEX 


Citric  acid,  production  of,  652 
Citromyces,  234,  653,  654 
citricus,  654 
Glaber,  654 
Pfefferianus,  654 
Citrus  canker,  973 
Cladosporium,  general  characters,  57 

herbarium,  57 

Cladothrix  dichotoma,  105,  312 
Classification  of  bacteria,  in 
Migula's,  112 
S.  A.  B.,  114 
of  enzymes,  208 
Claviceps  purpurea,  59 
Cleanliness  in  canning,  535 
Clostridium,  236 

americanum,  404 
Coagglutinins,  716 
Coagulating  basins,  324 

enzymes,  208,  213 
Cocci  in  milk,  451 

characteristics,  451 
Coccidiosis,  avian,  889 

of  rabbits,  889 
Coccidium,  137 
cuniculi,  138 

morphology,  889 
diseases  caused  by,  888 
hominis,  600 
schubergi,  18,  129 
Cockchafer,  septicaemia  of,  925 
Cocoanut,  bud  rot  of,  981 
Ccenocytes,  42 
Cold  as  preservative,  542 
Coleosporium   senecionis,    karyokinesis 

in,  29 

Colloidal  nature  of  soil,  349 
solution,  157,  179 
electrophoresis,  180 
emulsoid,  179 
gel,  1 80 
lyophilic,  179 
lyophobic,  179 
sol,  1 80 

suspensoid,  179 
Colloids,  177 


Colored  cheese,  499 
Colpidium  colpoda,  365 
Colpoda  cucullus,  in  soil,  365 
Colymbetes  fuscus,  919 
Commensals,  133 
Common  milk,  430 
Complement,  706 

anti-,  708 

deflection  of,  708 

deviation  of,  707 

fixation  of,  709 
Complementoid,  706 
Complementophile,  706 
Composition  of  beer,  622 

of  cell,  cell  contents,  186 

of  soil,  350 

Compressed  yeast,  559 
Concentrated  milk,  505 
Concentration,  hydrogen  ion,  162 

of  soils,  356 
Condensed  milk,  504,  531 

sweetened,  504 

unsweetened,  505 

Conductivity,  158  (See  also  ionization.) 
Conidia,  37 
Conidiophore,  37 
Conjugation,  in  protozoa,  131 
Conjunctiva,  692 
Contact  infection,  668 
Contagious  abortion,  810 

bovine  pleuro-pneumonia,  856 
Contents,  xv 
Control  of  cold  storage  industry,  549 

of  infectious  diseases,  754 
in  London,  Canada,  761 
practice  of,  756 
principles  of,  754 

of  preservation  of  food  by  chemi- 
cals, 558 

Copper  in  soil,  425 
Copra,  dried,  522 
Coprecipitins,  720 
Copula,  131 
Copulation,  130 
Corn,  canned,  534 

sterilization  of,  534 


INDEX 


IOOQ 


Counting  bacteria,  in  milk,  471 

in  soil,  370 
Cowpox,  857 
Cream,  cultures  for  ripening,  479 

ice,  512 

microorganisms  in,  476 

pasteurization  of,  530 

pasteurized,  481 

raw,  481 

ripening    (spontaneous    and    con- 
trolled), 477 

Crenothrix  polyspora,  109 
Crithidia,  characteristics,  135 
Crown  gall,  966 
Crystalloids,  158,  177,  178 
Ctenocephalis  felis,  946 
Cucumbers,  angular  leaf-spot  of,  974 
Cucurbits,  wilt  of,  988 
Culex  fatigans,  859 
Cultivation  of  bacteria,  118 

of  protozoa,  142 

of  yeasts,  76 
Cultures,  commercial,  479 

in  beer,  625 

in  leather,  656 

in  oleomargarine,  482 

in  pasteurized  cream,  481 

pure,  481 

in  raw  cream,  481 

in  vinegar,  641 

in  wine,  614 
Curd,  acid,  of  cheese,  486 

manipulation  of,  492 

rennet-,  of  cheese,  486 
Curdling  of  milk,  491 
Curing  by  chemical  preservation,  550 
Cutaneous  orifices,  protection  by,  688 
Cyanophyceae,  17,  21,  22,  94 

cellular  division  in,  30 

locomotion  in,  24 

membrane,  24 

nucleus  in,  97 

Cybister  laterimarginalis,  919 
Cycle  of  development,  99 
Cyst,  37 
Cytase,  208,  209 
64 


Cytology,  of  bacteria,  89 

elements  of,  15 

of  molds,  40 

of  yeasts,  63 
Cytolysins,  705,  709 
Cytophile  group,  706 
Cytoplasm,  appearance,  18 

of  bacteria,  89 

properties  of,  18 

of  protozoa,  125 
Cytotoxins,  705,  709 

anti-,  710 

auto-,  710 

leuco-,  710 

DASYHELEA  obscura,  945 
Decomposition  of  insoluble  food,  203 

type  of,  due  to  temperature,  272 

of  urea,  202 
Delhi  boil,  88 1 
Dematium,  62 

metachromatic  corpuscles  of,  44 

pullulans,  60 
morphology,  610 
in  slimy  wine,  604 
Dengue,  858 
Denitrification,  395 

environmental  relations,  396 
Dermatomycoses,  777 
Desiccation,  267 

of  foods,  516 
Determination  of  bacteria,  in  air,  305 

in  milk,  471 

in  soil,  371 

Development  of  bacteria  in  milk,  452 
Deviation  of  complement,  707 
Dialysis,  175 

Diarrhgea,  bacillary,  white,  806 
Diastase,  204,  208,  209 
Diffusion,  173 

in  cell,  184 
Digestive  tract,  microorganisms  of,  593 

study  of,  60 1 
Diphtheria,  antitoxin  for,  741 

fowl,  818 

human,  812-815 


IOIO 


INDEX 


Diphtheria,  Schick  test  for,  753 
toxin,  741 

toxin-antitoxin  mixture,  739 
Diphtheroids,  812 
Diplobacilli,  88 
Diplococcus,  87 

lymantriae  (See  bacillus.) 
melolonthae  (See  bacillus.) 
pneumoniae  (See  streptococcus.) 
Direct  division  of  nuclei,  34 
Direct  microscopic  count  of  milk,  471 
Dirt  in  milk,  466 
Diseases,  of  beer,  627 
contagious,  660 
control  of  infectious,  754 
due  to  food  poisoning,  587 
infectious,  659 
of  insects,  905 

American  foul  brood,  921 

European  foul  brood,  926 

flacherie  (silkworm),  907 

graphitosis,  920 

of  the  gut  epithelium,  929 

immunity,  946 

Japanese  gipsy  moth  disease,  909 

of  the  June  beetle  larvae,  906 

of  locusts,  912 

nosema-disease  of  bees,  939 

other  microbial  diseases,  945 

pathology,  946 

pebrine,  of  silkworm,  937 

pseudograsserie    of    gipsy    moth 

caterpillar,  930 
sacbrood  of  bees,  931 
septicaemia  of  caterpillars,  918 
septicaemia   of   cockchafer,    925, 

945 
septicaemia  of  larvae  of  Lamelli- 

cornae,  929 
wilt,  of  gipsy  moth  caterpillar, 

935 

of  man  and  animals,  775 
abscesses,  792 
actinobacillosis,  781 
actinomycosis,  778 
amoebic  dysentery,  876 


Diseases  of  man  and  animals,  anaplas- 

mosis,  898 
anthrax,  801-806 
Asiatic  cholera,  851-853 
aspergillosis,  775 
bacillary     white      diarrhoea     of 

chicks,  806 

balantidium  enteritis,  899 
barbers'  itch,  777 
beriberi,  591 
blackleg,  839 
boils,  792 

botryomycosis,  784 
botulism,  587 
bronchopneumonia,  795 
canine  distemper,  856 
cattle  plague,  856 
cerebro-spinal  meningitis,  787 
chicken  cholera,  807 

pox,  854 
cholera,  Asiatic,  851 

chicken,  807 

hog,  860 

chronic  bacterial  enteritis,  809 
coccidiosis,  889 
contagious  abortion,  810 
contagious   bovine   pleuro-pneu- 

monia,  856 
cowpox,  857 
Delhi  boil,  881 
dengue,  858 
dermatomycoses,  777 
diphtheria,  fowl,  818 

human,  812 
dourine,  888 
Dukes  disease,  855 
dysentery,  amoebic,  876 

bacillary,  816 
East  coast  fever,  896 
entero-hepatitis  of  turkeys,  879 
ergotism,  591 
erysipelas,  795 
favus,  778 

foot-and-mouth  disease,  859 
foot-rot  of  sheep,  838 
fowl  diphtheria,  818 


INDEX 


IOII 


Diseases   of    man    and    animals,    fowl 

plague,  860 
frambooesia,  904 
German  measles,  855 
glanders,  819 
gonorrhoea,  785 
hemorrhagic  septicaemia,  825 
herpes  tonsurans,  777 
hog  cholera,  860 
horsepox,  857 

sickness,  863 

human  trypanosomiases,  886 
infantile  paralysis,  864    • 
infectious  mastitis,  789 
influenza,  822 
kakke,  591 
kala  azar,  879,  880 
leishmaniasis,  88 1 
leprosy,  828 
lyssa,  868 
Madura  foot,  781 
malaria,  890 
mal  de  caderas,  887 
malignant  oedema,  839 
Malta  fever,  791 
measles,  855 
in  milk,  452,  468 
mumps,  854 
mycetoma,  781 
mycotic  lymphangitis,  782 
nagana,  887 
non-inheritance  of,  685 
organisms  classified,  660 
oroya  fever,  897 
osteomyelitis,  792 
pellagra,  591,  865 
plague,  830 
pneumomycoses,  775 
pneumonia,  798 
poliomyelitis,  864 
puerperal  septicaemia,  795 
pyaemia,  792 
pyorrhoea  aveolaris,  595 
rabies,  868 
red  water,  894 
relapsing  fever,  901 


Diseases  of  man  and  animals,  ring  worm, 

777 

scarlet  fever,  855 

septicaemia,  general,  795 

hemorrhagic,  825 

puerperal,  795 
sheep-pox,  857 
sleeping  sickness,  882 
smallpox,  854 

spirochaetal  diseases,  900,  904 
staphylococcic  infections,  792 
streptococcic  infections,  795 
surra,  887 
swamp  fever,  873 
swine  erysipelas,  832 
symptomatic  anthrax,  839 
syphilis,  903 
tetanus,  842 
Texas  fever,  894 
thrush,  776 
trichomycosis,  777 
trypanosomiases  of  animals,  887 

of  humans,  886 
tuberculosis,  833 
typhoid  fever,  845 
typhus  fever,  874 
white  diarrhoea,  bacillary,  806 
whooping  cough,  825 
wounds,  792 
yaws,  904 
yellow  fever,  875 
of  plants,  949 

blight  (stem)  of  alfalfa,  951 

of  beans  (bacteriosis),  953 

of  lettuce,  954 

of  mulberry,  955 

of  oats  (blade),  956 

of  pear,  958 

of  peas  (stem),  956 

of  sweet  peas  (streak),  962 

of  tomato,  963 

of  walnut,  963 
galls  and  tumors,  966 

crown  gall,  966 

finger  and  toes,  969 

olive  knot,  969 


1012 


INDEX 


Diseases  of  plants,  tuberculosis  of  sugar 

beet,  972 
leaf  spots,  973 

angular   leaf   spot   of   cucum- 
bers, 974 

citrus  canker,  973 

larkspur,  975 

plum  and  peach,  975 

sugar  beets,  976 
rots  (black),  of  cabbage,  978 

of  calla  lily  (soft  rot),  984 

of    carrots,    other   vegetables, 
984 

of  cauliflower,  983 

of  cocoanut  (bud  rot),  981 

of  hyacinth,  985 

of  musk  melon,  986 

of  orchids  (brown  rot),  982 

of  potatoes  (basal  stem  rot), 
980 

of  sugar  beets,  987 

Wakker's  hyacinth  disease,  979 
wilts,  of  cucurbits,  988 

egg  plant,  990 

Irish  potato,  990 

sweet  corn,  989 

tobacco,  990 

tomato,  990 
predisposition  to,  685 
protection  against  (See  protection), 

688 

transmitted  in  food,  582 
of  vinegar,  647 
of  wine,  608 
Disinfectants,  288 
acids,  292 
alcohols,  293 
alkalies,  292 
classification  of,  291 
essential  oils,  294 
formaldehyde,  294 
gases,  294 
hydrocarbons,  294 
mode  of  action,  288 
salts,  292 
Disinfection,  771 


Disinfection,  concurrent,  771 

curve  of,  289 

factors  influencing,  290 

terminal,  772 
Dispora  caucasica,  510 
Disposal  of  factory  refuse    (canning), 

540 

Dissemination  of  microorganisms,  677 
Dissociation  (See  ionization),  158 
Distribution  of  bacteria  in  soil,  368 
Division,  nuclear,  direct,  34 

general,  28 

indirect,  28 

in  molds,  43 
Dorset-Niles  (hog  cholera)  serum,   733, 

746 

Dourine,  888 
Droplet  infection,  665 
Drosophylla  cellaris,  648 
Drought  in  soil,  348 
Drying  of  foods,  516 

carbohydrates,  521 

fats,  522 

methods  of,  519 

proteins,  523 
Duke's  disease,  855 
Dust,  infection  from,  665 

influence  in  milk,  439,  443 
Dysentery,  amcebic,  876 

antiserum  for,  747 

bacillary,  816 
Dyticus  pisanus,  919 

EAST  coast  fever,  896 
Ectoplasm,  19 

protozoa,  125 
Egg  plant,  wilt  of,  990 
Eggs,  dried,  523 

refrigerated,  547 
Eimeria  avium,  138 

stiedae,  889 
Electrical  conductivity  (See  ionization), 

158 

Electricity,  influence  of,  282 
Electrolyte,  159 
Electrolytic  solution,  158 


INDEX 


1013 


Electrons,  158 

Elimination   of    microorganisms     from 

body,  678 

Emmenthaler  cheese,  500 
Empusa  aulicae,  918 

muscae,  944 
Emulsin,  205,  211 
Encystment  of  protozoa,  132 
Endo-enzymes,  208,  210,  214 
Endo-erepsin,  215 
Endomyces  decipiens,  42 
fibuliger,  41,  42 
magnusii,  41,  42,  43 
Endo-oxidase,  216 
Endoplasm,  19 

of  protozoa,  125 
Endospores,  84 

Endothelial    tissues,    effect    of    micro- 
organisms on,  682 
Endotoxins,  cause  of  infection,  676 
Endo-tryptase,  208,  215 
Energid,  15,  1 6 

in  molds, -41 
Energy,  155 

liberated  by  enzymes,  214 
for  metabolism,  200 
for  nutrition,  199 
source  of,  201 
Ensilage,  Monascus  in,  56 
Entamceba,  134 

buccalis,  594,  876 

morphology  of,  595 
coli,  661,  876 

agglutination  of,  716 
in  faeces,  599,  601 
morphology,  877 
dysenteriae,  in  intestine,  600 

isolation  from  fasces,  601 
gingivalis,  876 
histolytica,  18,  600 

in  amoebic  dysentery,  876 
morphology,  877 
meleagridis,  876 

in  entero-hepatitis  of  turkeys,  879 
tetragena,  66 1 
in  amoebic  dysentery,  876 


Enteritis,  balantidium,  899 

chronic  bacterial,  809 
Entero-hepatitis  of  turkeys,  879 
Entomogenous  fungi,  diseases  caused  by, 

944 
Entomophthoraceae,  diseases  caused  by, 

944 
Enzymes,  204,  248 

alcoholase,  208,  214 

amidase,  208,  215 

amylase,  208,  237 

autolytic,  208,  516- 

of  beer,  623 

of  carbohydrates,  208,  209 

casease,  493 

in  cells,  194 

cellulase,  208,  209 

chymosin,  208 

classification  of,  208 

coagulating,  208,  213 

cytase,  208,  209 

diastase,  204,  208,  209 

in  drying  of  foods,  516 

emulsin,  205,  211 

endo-,  208,  210,  214 

endo-erepsin,  215 

endo-oxidase,  216 

endo-tryptase,  208,  215 

energy  liberated  by,  214 

erepsin,  208 

ereptase,  204,  208,  212 

exo-,  208 

of  fat,  208,  2ii 

of  fermentation,  207 

galactase,  494 

general,  219 

hydrolyzing,  208,  209 

invertase,  204,  205,  208,  210,  214 

katalase,  208,  216 

lactacidase,  208,  214 

lactase,  208,  210,  214 

lipase,  204,  208,  211,  214 

maltase,  208,  210,  214 

oxidase,  216 

oxidase  on  grapes,  605 

oxidizing,  208,  216 


INDEX 


Enzymes,   pepsin,    204,    208,   212,    214, 
491,  494 

peptase,  208 

peroxidase,  216 

properties  of,  205 

proteases,  208,  211 

proteolytic,  208,  211 
action  upon  albumin,  egg,  fibrin, 

gelatin,  milk,  serum,  213 
determination  of,  212 
of  yeasts,  215 

I  ^i    carrot* 

pyocyanase,  300 

reducing,  208,  216 

reductases,  208,  216 

rennet,  205,  208,  213 

rennin,  491 

reversibility  of  action,  218 

soluble,  214 

steapsin,  208,  211 

sucrase,  210 

thrombase,  208,  213 

trypsin,  204,  208,  212,  214 

tryptase,  208 

tyrosinase,  208,  216 

urease,  208,  214 

vinegar-oxidase,  208,  214,  216 

in  yeast  preparation,  560 

of  yeasts,  215 

zymase,  207,  208,  214 
Enzymic  theory  of  anabolism,  217 

of  katabolism,  217 
Epiplasm,  27,  45 

of  yeasts,  69 

Epithelial    tissues,    action    of    micro- 
organisms on,  682 
Epitoxoid,  704 

Equations  of  fermentations,  230 
Equatorial  plates,  28 
Erepsin,  208 
Ereptase,  204,  208,  212 
Ergotism,  591 
Erysipelas,  795 

of  swine,  832 

Erythrocytes,  effect  of  microorganisms 
on,  682 


Eubacteria,  79 

Euglena  splendens,  protomitosis  of,  31 

viridis,  21 

Euproctis  chrysorrhea,  931,  945 
European  foul  brood,  926 
Evaporation  of  foods,  516 

of  milk,  505 

Exhaustion  theory  of  immunity,  721 
Exo-enzymes,  208 
Extracellular  fermentation,  203 

FACTORY  refuse  (canning),   disposal 

of,  540 
Facultative  aerobes,  228 

anaerobes,  228 

parasites,  132,  660 

saprophytes,  660 
Faeces,  microorganisms  of,  597 
Fat,  in  cells,  191 

enzymes  of,  208,  211 

fermentation  of,  239 

in  soils,  378 
Fatty  foods,  drying  of,  522 

preservation  of,  522 
Favus,  778 

Feeding,  influence  on  milk,  443 
Fermentation,  abnormal  of  milk,  463 

acetic,  235,  636 

of  acetic  acid,  649 

of  acetone,  649 

acid-gas,  235 

of  alcohol,  634 

alcoholic,  225,  231,  234 
equation  of,  231 
in  milk,  466 

of  amygdalin,  211 

of  beer,  625 

of  bread,  564 

of  butter,  483 

butter-milk,  512 

butyric,  232,  236,  612 

of  canned  foods,  540 

carbon  dioxide,  199 

cellulose,  237,  333,  362,  375 

of  cheese,  492 

of  cider,  629 


INDEX 


1015 


Fermentation,  citric,  234,  653 
curdled  milk,  acid,  462,  491 

sweet,  464 

of  dextrose,  225,  231,  235 
of  disaccharides,  237 
energy  of,  214 
enzymes  of,  207 
equations  of,  230 
extracellular,  203 
of  fats,  211,  239,  334,  378 
of  flax,  658 
of  ginger  beer,  63 1 
glycerin,  239 
hippuric  acid,  244 
hydromel,  629 
indigo,  658 
intracellular,  203 
kefir,  508 
kelp,  650  9 

koji,  630 
kumyss,  508 
lactic,  225,  232,  235,  236 
lactic  acid,  651 
of  lactose,  232 
of  leather,  655 
of  leben,  510 
of  malt  syrups,  577 
of  mannit,  611 
mead,  629 
mechanism  of,  203 
Mexican  pulque,  630 
of  milk,  460 
molds  of,  50,  56 
moto,  630 
of  olives,  572 
organic  matter,  345 
perry,  628 
pickles,  571 
pombe,  631 
of  protein,  332,  380 
proteolytic,  205,  239,  332,  380 
ragi,  635 
rice  beer,  630 
of  saccharose,  237 
of  sake,  630 
of  sauerkraut,  570 


Fermentation  of  sewage,  332 

of  silage,  574 

of  starch,  209,  237,  378 

of  starch  manufacture,  579 

of  sugar,  209,  225,  233,  378 

in  sugar  manufacture,  580 

of  tanning,  655 

of  tea,  580 

of  tobacco,  578 

of  urea,  243 

of  vinegar,  640 

of  waxes,  378 

of  white  lead,  654 

of  wine,  603 

of  yahourth,  510 

of  yeast  manufacture,  560 

yeasts  of,  73 
Fever,  East  Coast,  896 

Malta,  791 

oroya,  897 

relapsing,  901 

swamp,  873 

of  Texas  cattle,  894 

typhoid,  845 

typhus,  874 

yellow,  875 
Filters,  life  in,  339 

porous,  325 

sand,  323 

sewage,  341 

Filtrable  microorganisms  (viruses),  119, 
66 1 

cultivation  of,  121 

determination  of,  122 

evidences  of,  119 

of  flacherie,  935 

of  sacbrood,  931 
Filtrate,  blackleg,  731 
Filtration  of  water,  323, 324, 325 
Fingers-and-toes,  969 
Fire  blight,  959 
Fish,  chemical  preservation  of,  551 

dried,  523 

poisoning,  585 

refrigeration  of,  544 

sterilization  of,  534 


ioi6 


INDEX 


Fixation  of  atmospheric  nitrogen,  400 

of  complement,  709 
Flacherie    of    gipsy-moth     caterpillar, 

935 

of  silkworm,  906 
Flagella,  24 

of  bacteria,  105 
formation  of,  25 
lophotrichous,  105,  106 
monotrichous,  105,  106 
peritrichous,  105,  106 
of  protozoa,  127 
Flagella ta,  characteristics,  134 
diseases  caused  by,  879 
locomotion  in,  24 
Food,  alteration  by  heat,  526 
appearance  changes,  527 

due  to  heat,  526 
B.  botulinus  in,  587 
changes  after  cold  storage,  544 

chemical  from  heating,  527 

during  chilling,  543 

during  cold  storage,  543 
desiccation  of,  516 
detection  of  spoilage,  540 
digestibility  changes  in,  528 
drying,  516 
evaporation  of,  516 
effect  of  preservatives,  550 
fermented,  559 
home  canning  of,  539 
infection  from,  666 
mechanical  disintegration  by  heat, 

p7 

of  microorganisms,  195,  221 
alcohol,  224 
amino-acids,  223 
ammonia,  222,  223 
amount  required,  221 
asparagin,  223 
carbohydrates,  223,  226 
carbon  dioxide,  222,  223,  226 
carbon  monoxide,  222,  224 
for  energy,  224 
for  growth,  222 


Food  of  microorganisms,  hydrogen,  222, 

224,  226 

hydrogen  sulphide,  224 
methane,  222,  224,  226 
methods  of  securing,  195 
minerals,  222,  224 
nitrates,  223,  226 
nitrites,  224 
nitrogen  (free),  223 
organic  acids,  223,  226 
oxygen,  223,  226 
peptones,  223 
in  soil,  358 
thiosulphate,  224 
normal  fauna  of,  528 
normal  flora  of,  528 
palatability  changes  by  heat,  528 
pasteurization  of,  530 
poisoning,  581 
preservation  by  chemicals,  550 

by  cold,  542 
processing  of,  532 
spoilage  of  canned,  540 
sterilization  of,  532 
yeast  as,  563 

Foot-and-mouth  disease,  859 
Foot  rot  of  sheep,  838 
Formaldehyde,  as  disinfectant,  294 

as  preservative,  557 
Formic  acid  as  preservative,  556 
Foul  brood,  American,  920 

European,  926 
Fowl  diphtheria,  818 

plague,  860 
Frambooesia,  904 
Freezing  of  organisms,  274 
Fruits,  chemical  preservation  of,  553 
pasteurization  of  juices,  530 
refrigeration  of,  548 
sterilization  of,  535 
Fungi,  basis  of  classification,  37 
general,  36 
imperfect,  40,  6r 
reproduction  of,  37 
Fusarium,  general  characters  of,  57 


INDEX 


1017 


GALACTIMA  succosa,  mesomitosis  of, 

33 

Galleria  melonella,  946 
Gallionella  ferruginea,  no 
Galls,  crown,  966 
Gametocytes,  of  protozoa,  131 
Gas  gangrene,  antiserum  for,  745 
Gassy  cheese,  498 

fermentation  of  milk,  463 
Gelatin,  523 
Gemma,  37 
Gemmulation,  27 

of  protozoa,  128 
Genito-urinary  tract,  692 
Geotropism,  284 
German  measles,  855 
Germicidal  action  of  milk,  461 
Germination  of  ascospores,  67 

of  spores,  86 
Ghee,  522 
Ginger  beer,  631  • 

Gipsy  moth  caterpillar,  parasitic  bac- 
teria of,  945 

pseudograsserie  of,  930 

wilt  disease  of,  935 
Gipsy     moth,     Japanese    disease     of, 

909 
Glanders,  819 

agglutination  test  for,  752 

mallein  test  for,  750 
Glaucoma  piriformis,  19 
Glossina  morsitans,  882,  887 

palpalis,  882 

Glycogen,  as  reserve  product,  22 
Gonorrhoea,  785 

antiserum  for,  746 
Gorgonzola  cheese,  502 
Gortyna  ochracea,  946 
Granulobacter  pectinovorum,  658 
Graphitosis,  920 
Gravity,  influence  of,  284 
Gregarinae,  137 

carbohydrate  in,  190 
Growth,  inhibition  of,  288 

stimulation  of,  286 
Gryllus  pennsylvanicus,  917 


Guaranteed  milk,  432 
Gymnoasceae,  61 

ILEMOGREGARIN^E,  139 
Haemoproteus,  139 
Haemorrhagic  septicaemia,  825 
Haemosporidia,  138 

pathogenic,  890 
Hail,  microorganisms  in,  317 
Hanneton  commun,  946 

de  la  Saint- Jean,  946 
Haplosporidia,  139 

parasitic,  898 
Haptophile  receptors,  700 
Haptophore  group,  700 
Heat  as  preservative,  524 

production,  250 

required  for  canning,  536 
Heliotaxis,  280 
Heliotropism,  280 
Hemoagglutinins,  717 
Hemolysins,  705 

normal,  696 
Hemoopsonins,  714 
Hepatozoo'n  perniciosum,  139 
Herpes  tonsurans,  777 
Herpetomonas,  characteristics,  135 

muscae-domesticae,  134 
Heterogamy,  34 
Heterolysins,  696 
Hetero trophic  organisms,  223 
Higher  bacteria,  107 

plants  in  soil,  367 
Hippuric  acid,  production  of,  243 
Histoplasma  capsulatum,  139,  903 
History  of  canning,  524 

of  microbiology,  i 

of  non-symbiotic  nitrogen  fixation, 
401 

of  symbiotic  nitrogen  fixation,  405 
Hog  cholera,  860 

antiserum  for,  733,  746 
Holophytic  organisms,  196 
Holozoic  organisms,  196 
Home  canning  of  foods,  539 
Hormodendron,  57 


ioi8 


INDEX 


Hormones,  147 
Horse  pox,  857 
Horse  sickness,  863 
Hyacinth,  soft  rot  of,  985 

Wakker's  disease,  979 
Hydrogen,  displaceable,  160 

as  food,  222,  224,  226 

oxidation  in  soil,  351,  377 

production  in  soil,  375 

sulphide  as  food,  224 
Hydrogen  ion  concentration,  determina- 
tion of,  165 

of  soils,  356 

theory  of,  162 

Hydrolyzing  enzymes,  208,  209 
Hydromel,  629 
Hydrophilus  pistaceus,  919 
Hyperimmune  serum,  746 
Hypha,  36 
Hyphomycetes,  251 
Hyposulphite  oxidation,  201 

ICE  CREAM,  512 

increase  of  bacteria  during  storage, 

5H 

poisoning,  586 
Immunity,  acquired,  687,  697,  725 

active,  697,  725 

definition  of,  684 

exhaustion  theory  of,  721 

familial,  688 

general,  684 

individual,  688 

of  insects,  946 

natural,  687,  725 
factors  of,  688 

noxious  retention  theory  of,  721 

passive,  697,  698,  725 

phagocytic  theory  of,  723 

plants,  950 

racial,  687 

side  chain  theory  of,  722 

theories  of,  721 
Imperfect  fungi,  40,  61 
Index,  opsonic,  712 

percentage,  713 


Index,  phagocytic,  712 
Indigo,  658 

Indirect  division  of  nucleus,  28 
Indol,  production  of,  241 
Infantile  kala  azar,  880 

paralysis,  864 

control  of,  in  London,  Canada, 

763 
Infection,  air-borne,  664 

animal  carriers  of,  667 

by  biological  agents,  773 

cause  of,  675 

contact,  668 

defined,  659 

droplet,  665 

dust,  665 

factors  influencing,  674 

food,  581,  666 

human  carriers,  668 

manner  of  entering  body,  664 

milk-borne,  467,  666 

number  of  organisms  involved    in, 
674 

resistance  against,  675 

routes  of,  669,  754 

soil,  666 

sources  of,  664 

staphylococcic,  792 

streptococcic,  795 

variations  of,  672 

virulence  of,  674 

water-borne,  666 
Infectious  mastitis,  789 
Inflammatory  processes,  693 
Influenza,  822 

Influenza-pneumonia  vaccine,  738 
Infusoria,  characteristics,  139 

parasitic,  899 
Inheritance  of  disease,  685 
Inorganic  food  preservatives,  555 
Insects,  diseases  of,  905 

immunity  in,  946 

pathology  of,  946 
Inspected  milk,  432 
Intercellular  relations,  300 
Intestine,  microorganisms  of,  596 


INDEX 


IOIQ 


Intestine,  protection  by,  691 
Intracellular  fermentation,  203 
Invertase,  204,  205,  208,  210,  214 
Involution  forms,  80 
lodococcus  magnus,  in  mouth,  594 

parvus,  in  mouth,  594 

vaginatus,  in  mouth,  594 
Ion,  159 
lonization,  158 
t        anion,  159,  160 

anode,  160 

atoms,  158 

cathode,  160 

cations,  159,  160 

displaceable  hydrogen,  160 
f       electron,  158 

valence  electron,  158 
Iron  in  soil,  425 
Isinglass,  in  fining  wine,  620 
Isogamous  fertilization,  131 
Isogamy,  34,  131 
Isohemoopsonin,  714 
Isolysin,  696 
Isoprecipitin,  719 

JAMS,  preservation  of,  522 
Japanese  gipsy  moth  disease,  909 
Jellies,  preservation  of,  522 
June  beetle  larvae,  bacterial  disease  of, 
906 

KAKKE,  591 
Kala  azar,  879 

infantile,  880 
Karyokinesis,  28 

in  bacteria,  89 

in  molds,  43 

in  protozoa,  130 

in  yeasts,  68 
Karyosome,  17 
Katabolism,  200,  203 

enzymic  theory  of,  217 
Katalase,  208,  216 
Kefir,  508 

grain  of,  513 
Kelp,  650 


Kinds  of  cheese,  500 
Kinetonucleus,  18 
Koji,  630 
Kumyss,  508 

LACHNOSTERNA  spp.,  906 
Lactacidase,  208,  214 
Lactase,  208,  210,  214 
Lactic  acid,  production  of,  651 

bacteria  in  milk,  446 
in  wine,  610 

fermentation,  235 

equation  of,  225,  232 
Lager  beer,  622 
Lamblia,  characteristics,  136 

intestinalis,  137,  600 
in  faeces,  599 
morphology,  136 
Lamellicorna,  920 

septicaemia  of  larvae  of,  929 
Larkspur,  leaf  spot  of,  975 
Laverania  malariae,  892 

sporozoits  of,  138 
Leather,  manufacture  of,  655 

pure  culture  for,  656 
Leben,  510 
Leishmania,  disease  producing,  879 

donovani,  900 
morphology,  880 

infantum,  880 

tropica,  in  Delhi  boil,  88 1 
Leishmaniasis,  localized,  88 1 
Leochracea,  no 
Leprosy,  828 
Leptospira  icteriodes,  in  yellow  fever, 

876 
Leptothrix  buccalis,  in  mouth,  594 

ochracea,  no 
Lettuce,  blight  of,  954 
Leucin,  241 

Leucocytes,  effect  of  microorganisms  on, 
682 

in  milk,  471 
Leucocytotoxin,  710 
Leucocytozoon  lovati,  spore  formation 
in,  27 


IO2O 


INDEX 


Leuconostoc  mesenterioides,  522 
Leucoplastids,  20 
Life  cycle  of  protozoa,  131 
Light,  heliotaxis,  280 

heliotropism,  280 

influence  of,  278 

upon  organisms  in  water,  319 

phototaxis,  280 

phototropism,  280 

production,  251 

radium  rays,  281 

X-rays,  281 
Linin,  16 

of  protozoa,  1 26 
Lipase,  204,  208,  211,  214 
Locomotion,  24 

protozoa,  127 

Locusts,  bacterial  disease  of,  912 
Luetin,  753 
Lug    worm,    bacterial    disease    of    gut 

epithelium,  929 
Lungs,  protection  by,  691 
Lymantria  dispar,  930,  946 
Lymphangitis,  mycotic,  782 
Lysin,  704 

bacterio-,  705 

cyto-,  705 

hemo-,  705 

hetero-,  696 

iso-,  696 

-    structure  of,  706 
Lysinogen,  706 
Lyssa,  868 

MACARONI,  522 
Macrogametes,  in  protozoa,  131 

in  yeasts,  70 
Macronucleus,  18 
Madura  foot,  781 
Magnesia  in  soil,  417 
Mai  de  caderas,  887 
Malacosoma  neustria,  946 
Malaria,  890 
Malignant  oedema,  839 
Mallein,  750 
Malt  syrups,  577 


Malta  fever,  791 
Maltase,  208,  210,  214 
Malting,  560 

of  beer,  623 
Manganese  in  soil,  425 
Mannitic  bacteria,  in  wine,  611 
Market  milk,  430 
Mastitis,  infectious,  789 
Matzoon,  510 
Mead,  629 
Measles,  855 
Meat,  chemical  preservation  of,  551 

jerked,  523 

poisoning,  584 

refrigeration  of,  544 

sterilization  of,  533 
Mechanical  effects,  influence  of,  283 
Mechanism  of  cells,  151 
Media,  grape  juice  as  culture,  603 

soil  as  culture,  346 

wine  as  culture,  603 
Melolontha  vulgaris,  918,  925 
Membrane,  23 

impermeable,  175 

permeable,  175 

semi-permeable,  175 

undulating,  25,  127 

Meningitis,  epidemic  cerebrospinal,  787- 
789 

antiserum  for,  745 

control   of,   in  London,    Canada, 

763 

Mercaptan,  242 
Merismopedia,  87 
Merozoites,  129 
Mesomitosis,  32 

in  protozoa,  130 

in  yeasts,  69 
Metabiosis,  298 
Metabolism,  195,  200,  203 

mechanism  of,  203 

products  of,  233 
physical,  250 

of  protozoa,  198 

relation  of  infectious  organisms  to, 
681 


INDEX 


1021 


Metachromatic  corpuscles,  20,  22 

in  bacteria,  90,  101 

in  molds,  44 

in  yeasts,  64,  67 
Metachromatin,  45 
Metachromatism  in  molds,  44 
Metamitosis,  30 
Metaphase,  28 
Methane,  as  food,  222,  224,  226 

oxidation  in  soil,  377 

production  in  soil,  375 
Methods  of  attenuating  viruses,  726 

of  control  of  infectious  diseases,  756 

of     counting     microorganisms    in 
milk,  471 

of  disinfection,  771 

of  infection,  659 

of  study  of  bacteria  in  digestive 
tract,  601 

of  study  of  bacteria  in  soil,  370 
Mexican  pulque,  630 
Microbial  content  of  milk,  429 

diseases  of  plants,  949 

food  poisoning,  581 
Microbiology,  of  acetic  acid,  649 

of  acetone,  649 

of  air,  303 

of  alcohol,  distilled,  631 

of  antisera,  740 

of  beer,  622 

of  bread,  564 

of  butter,  474 

of  cheese,  486 

of  cider,  628 

of  citric  acid,  652 

of  compressed  yeast,  559 

of  dairy  (various)  products,  504 

of  diseases  of  insects,  905 

of  fermented  foods,  559 

of  food  poisoning,  581 

of  foods    preserved    by    chemicals, 

550 

by  cold,  542 
by  drying,  516 
by  heat,  524 

of  ginger  beer,  631 


Microbiology,  history  of,  i 

of  human  and  animal  diseases,  659, 

775 

of  hydromel,  629 

of  immunity,  684 

of  indigo,  658 

of  lactic  acid  production,  651 

of  leather,  655 

of  malt  syrups,  577 

of  mead,  629 

of  Mexican  pulque,  630 

of  milk,  428 

of  olive  pickling,  572 

of  perry,  628 

of  pickles,  571 

of  plant  diseases,  949 

of  pombe,  631 

of  retting,  658 

of  rice  beer,  630 

of  sake,  630 

of  sauerkraut,  570 

scope  of,  1 1 

of  sewage,  330 

of  silage,  574 

of  soil,  345 

of  special  industries,  649 

of  starch  production,  579 

of  sugar  production,  580 

of  susceptibility,  684 

of  tanning,  655 

of  tea,  580 

of  tobacco,  578 

of  vaccines,  724 

of  vinegar,  636 

of  white  lead,  654 

of  wine,  603 
Micrococcus  aquatilis,  312 

ascoformans,  784 

candicans,  312 

coronatus,  312 

epidermidis  albus,  in  purulent  in- 
flammations, 794 

gonorrhceae,  for  antiserum  produc- 
tion, 746 

in  conjunctiva,  670 
in  genital  tract,  671,  692 


IO22 


INDEX 


Micrococcus  gonorrhoea  in  gonorrhoea, 

785 

morphology,  786 

vaccine,  737 
intracellularis  meningitidis,  662 

in  cerebrospinal  meningitis,  787 

in  eye,  670 

in  healthy  persons,  663 

immunization  with,  745 

morphology  of,  788 

in  nasal  cavity,  690 
melitensis,  in  Malta  fever,  791 

morphology  of,  791 
nigrofaciens,  906 

morphology  of,  906-907 
nivalis,  312 

pneumoniae  (See  streptococcus.) 
progrediens,  size  of,  121 
pyogenes,  in  botryomycosis,  784 

in  healthy  persons,  663 
pyogenes  var.  albus,  in  healthy  per- 
sons, 688 

in  mouth,  594 

in  nasal  cavity,  690 

in  purulent  inflammations,  794 

vaccine,  737 

pyogenes  var.  aureus,  agglutinins  in 
blood,  714 

effect  of  pressure  on,  283 

in  genital  tract,  672 

in  healthy  persons,  688 

hemolysis  of,  682 

morphology,  792 

in  mouth,  594 

in  nasal  cavity,  690 

proteolytic  enzyme  of,  213 

in  purulent  inflammations,  792 

toxin  of,  676 

vaccine,  737 

pyogenes  var.  citreus,  in  purulent 
inflammations,  794 

vaccine,  737 
Microgametes,  in  protozoa,  131 

in  yeasts,  70 

Microglena  punctifera,  21 
Micronucleus,  18 


Microorganisms,  in  acetone  production, 

650 
acetic  in  vinegar,  636 

in  wine,  609 

acid-forming  in  milk,  444 
aerobic,  in  soil,  228,  350 

in  wine,  608 
air,  303 
of  alcohol,  635 
algae  in  soil,  364 
anaerobic,  in  sewage,  228,  332 

in  soil,  350 

in  wine,  609 
autotrophic,  223 
B.  coli  in  water,  313 

coli-aerogenes  in  milk,  448 

enteritidis  sporogenes  in  water, 

3i3 

typhosus  in  milk,  468 

in  water,  314 
Bact.  bulgaricum  in  milk,  450 

lactis  acidi  in  milk,  446 

lactis  aerogenes  in  water,  314 
of  beer,  624 

in  body  of  sound  individuals,  663 
of  brewing,  622 
of  cheese,  486 

chromoparous  bacteria,  246 
chromophorous  bacteria,  246 
of  cider,  628 
of  citric  acid,  654 
classes  in  water,  311 
cocci  in  milk,  451 
complexity  in  sewage,  330 
of  compressed  yeast,  560 
control  of,  in  wine,  612 
cosmopolitan  saprophytes,  47 
of  cream,  476 
in  dairy  utensils,  440 
decrease  in  water.  319 
determination  in  air,  305 

in  milk,  471 
in  digestive  tract,  593 

methods  of  study  of,  601 
of  disease,  660 

of  insect  diseases,  905 


INDEX 


1023 


Microorganisms,  dissemination  of,  677 
effect  on  body,  680 
elimination  from  body,  678 
entrance  into  air,  304 
facultative  anaerobic,  228 
of  faeces,  597 
filtrable,  119,  661 
food  of,  221 
in  food,  516 
in  food  poisoning,  584 
on  grapes,  604 
heterotrophic,  223 
holophytic,  196 
holozoic,  196 
of  intestine,  596 
kinds  in  air,  303,  307 

in  canned  food,  528 

in  cream,  476 

in  milk,  444 

in  sewage,  330 

in  soil,  350,  360 

in  water,  311 
in  lactic  acid,  651 
of  leather,  655 
metabolism  of,  195 
methods  of  study  in  soil,  370 
Microspira  comma  in  water,  314 
in  milk,  430 

certified,  432 

special,  432 
molds  in  soils,  360 
morphological  groups  in  soil,  370 
of  mouth,  593 
nitric,  390 
nitrous,  390 

normal  development  in  milk,  460 
number  in  air,  306 

in  concentrated  milk,  506 

in  cream,  476 

in  ice  cream,  513 

injnilk,  429 

in  soil,  367 
nutrition  of,  195 
obligate  aerobic,  228 
obligate  anaerobic,  228 
occurrence  in  air,  304 


Microorganisms  in  olives,  573 
oxygen  relations,  225 
parachrome  bacteria,  246 
parasitic,  196 
pathogenic,  338 

in  butter,  485 

in  milk,  452,  468 
period  of  incubation  in  body,  680 
photogenic,  251 

physiological  group  in  soil,  370 
physiology  of,  145 
of  plant  diseases,  949 
products  of  activities,  230 
propionic,  610 
prototrophic,  223 
protozoa  in  soil,  364 
putrefactive  in  sewage,  332 
in  rain,  316 
reactions  on  body,  680 
of  retting,  658 
in  rivers,  318 
saprophytic,  196 
saprozoic,  196 
in  sauerkraut,  570 
in  sea  water,  318 
in  sewage,  330 
sewage  streptococci,  313 
of  silage,  576 

slime-forming  in  wine,  610 
in  snow,  317 
in  soil,  367 
in  soil  fertility,  345 
soil  bacteria  in  water,  312 
sources  in  milk,  434 
spores  in  canning,  536 
of  stomach,  596 
subsidence  in  air,  305 
in  sugar  production,  580 
thermal  death-point  of,  275 
thermophilic,  271 
typical  forms  in  sewage,  330 
in  udder  of  cow,  434 
in  upland  waters,  318 
of  vinegar,  636 
in  water,  310 
in  wells,  317 


1024 


INDEX 


Microorganisms  in  wine,  608 
Microspira  aestuarii,  424 
comma,  705 

agglutinins  in  blood,  697,  714,  715 

ash  elements  in,  193,  194 

effect  of  pressure  on,  284 

facultative  saprophyte,  660 

flagella,  25,  105 

in  intestine,  600,  692 

morphology,  851-852 

precipitins  for,  718 

production  of  vaccine,  738 

protein  content  of,  190 

proteolytic  enzyme  of,  213 

toxin  of,  676 

variation  in  infection,  673 

in  water,  314 
Microsporidia,  139,  938 

parasitic,  899 

Milk,  acid  forming  bacteria  in,  444 
aeration  of,  455 
alcoholic,  466 
analysis  of,  470 
B.  coli  aerogenes  in,  448 
Bact.  bulgaricum  in,  450 
Bact,  lactis  acidi  in,  446 
bitter,  465 
butter-,  446 

centrifugal  separation  of,  456 
certified,  432 

Boston,  433 

Brooklyn,  433 

Chicago,  433 

New  York  City,  433 
changes  due  to  organisms,  429 
character  of,  428 
chemicals    in    checking    bacteria, 

"460 

cocci  in,  451 
common,  430 

Boston,  430 

Chicago,  430 

Connecticut  cities,  430 

Ithaca,  431 

Montclair,  431 

Rochester,  469 


Milk,  condensed,  504 

concentrated,  505,  506 

powdered,  507 

sweetened,  504 

unsweetened,  505 
contamination  of,  441,  452 
curdling  by  acid,  462 

by  rennin,  491 

development  of  bacteria  in,  452 
dirt  in,  466 
disease  carrier,  467 
dried,  523 
drinks,  507 

butter-milk,  512 

kefir,  508 

kumyss,  508 

leben,  510 

matzoon,  510 

yahourth,  510 
dust,  influence  of,  439,  443 
evaporated,  505 
exterior  of  cow's  body  in  relation  to, 

438 
feeding,  influence  on  germ  content, 

443 

fermentation  (abnormal)  of,  463 
fermentation  (normal)  of,  460 
gassy  fermentation  of,  463 
germicidal  action,  461 
guaranteed,  432 
house,  439 
inspected,  432 
kinds  of  organisms  in,  444 
leucocytes  in,  471 
market,  430 

microbial  content  of,  429 
microscopic    method    of    analyses, 

47i 

milker  in  relation  to,  439 
neutral  bacteria,  451,  468 
odors,  428 
pail,  442 

pasteurization  of,  459,  530 
pathogenic  bacteria  in,  452,  468 
periods  of  change,  461 
plating  method  of  analysis,  471 


INDEX 

• 


1025 


Milk,  poisonous,  586 

powdered,  507 

proteolytic  bacteria  in,  451 

proteolytic  changes  in,  451 

quality  for  cheese  making,  487 

refrigeration,  547 

ripening  of,  490 

ropy,  464 

selected,  432 

slimy,  464. 

sources  of  organisms  in,  434 

special,  432 

standards  of,  471 

straining  of,  453 

sweet  curdling  of,  464 

taints,  428 

temperature,    influence    on    germ 
content,  458 

tests  for  quality,  489 

in  udder  of  cow,  434 

utensils  for,  440,  444 

value    of  standards  and  analyses, 
472 

water  supply  in  relation  to,  441 
Milker  in  relation  to  milk,  439 
Milk-house,  air  of,  439 
Mineralization  in  soil,  351 
Minerals,  as  food,  222,  224,  358 

oxidation  of,  244 

reduction  of,  244 
Mitochondria,  18,  19 

granular,  19 

in  molds,  42 

rod-shaped,  19 

thread-like,  19 
Mitosis,  28 

of  protozoa,  130 

in  yeasts,  68 
Moisture,  in  cells,  187 

influence  of,  263 

requirement,  221 

in  soil,  346 
Molasses,  522 
Molds,  ash  elements  in,  193 

in  canned  foods,  528 

cell  wall,  47 
65 


Molds  on  cheese,  499 

cytology  of,  40 

cytoplasm  of,  42 

diseases  caused  by,  775 

of  fermentation,  48,  50,  56 

on  foods,  518 

genera  of,  48 

general  structure  of,  40 

on  grapes,  604 

metachromatic  corpuscles  of,  44 

nucleus  of,  42 

parasitic,  48 

reserve  products  of,  44 

saprophytic,  47 

in  soil,  360 

structure,  36 

Molluscum  contagiosum,  900 
Monas,  135,  365 

termo,  26 
Monascus  in  ensilage,  56 

purpureus,  56 
Monilia,  59 

Candida,  59 

sitophila,  59 

Monosporella  unicuspidata,  945 
Morphological    groups    of    bacteria    in 

soil,  369 
Morphology,  bacteria,  79 

molds,  36 

protozoa,  124 

yeasts,  61 
Motility,  bacteria,  81 

protozoa,  127 

spirochaetae,  107 
Moto,  630 
Mouth,  microorganisms,  593 

protection  by,  690 
Mucor,  48 

circinelloides,  41,  51 

general  characters,  48 

javanicus,  51 

mucedo,  50 

oryzae,  633,  635 

plumbeus,  51 

racemosus,  50 

rouxii,  50,  633 


IO26 


INDEX 


Mucor,  stolonifer,  49 

Mucous  membranes,  protection  by,  689 

Mulberry,  blight  of,  955 

Mumps,  854 

Muskmelon,  soft  rot  of,  986 

Must  of  grapes,  603 

Mutual  influences,  297 

Mycelium,  36 

Mycetoma,  781 

Mycetozoa,  124 

Mycoderma,  61,  238 

aceti,  in  vinegar,  637 
cerevisiae,  in  yeast,  563 
vini,  77 

in  fermented  foods,  570 

on  grapes,  608 

in  pickles,  571 

in  vinegar,  637 

in  wine,  609 

in  yeast,  563 
in  wine,  76 

Mycorrhiza,  in  soil,  363 
Mycotic  lymphangitis,  782-784 
Myonemes,  127 
Myxosporidia,  139 
parasitic,  899 

NAGANA,  887 

Nasal  cavity,  protection  by,  690 
Nasturtium,  spot  of  leaves,  976 
Natural  immunity  in  plants,  950 
Negri  bodies,  870 

staining  of,  871 
Neurin,  241 
Neutral  spirits,  63  2 
Neutrality,  161 
Nipa  cinerea,  919 
Nitrates,  accumulation  of,  394 

disappearance  of,  394 

as  food,  223,  226 

reduction  in  sewage,  335 
Nitric  acid,  as  preservative,  555 
Nitrification,  389 

accumulation  of  nitrates,  394 

disappearance  of  nitrates,  394 

environmental  relations,  391 


Nitrification,  experimental  study,  389 

nitric  bacteria,  390 

nitrous  bacteria,  390 
Nitrite,  as  food,  224 

reduction,  337 

in  sewage,  337 
Nitrobacter,  391 

in  sewage,  337 
Nitrogen,  addition  to  soil,  374 

in  cells,  188 

cycle,  260 

as  food,  223 

oxidation  in  soil,  351 

source  in  soil,  400 

utilization  by  molds  in  soil,  362 
Nitrogen  fixation  (atmospheric),  400 

aerobic  species,  402 

anaerobic  species,  402 

cultures  for  inoculation,  414 

development  of  organisms,  408 

energy  relations,  404 

entrance  of  organisms,  408 

environmental  relations,  410 

inoculations,  methods  of,  412,  414 

mechanism,  410 

by  molds  in  soil,  362 

non-symbiotic,  401 

physiological  efficiency,  409 

resistance  of  plants  to,  409 

specialization  of  organisms,  410 

symbiotic,  405 

theories  of,  400 

variations  of,  410 
Nitrosococcus,  391 
Nitrosomonas,  391 

oxidation  by,  201 

in  sewage,  337 

Nitrous  acid,  as  preservative,  555 
Noctiluca,  phosphorescence  of,  251 

miharis,  nitrogen  content,  189 
Nomenclature,  117 
Non-inheritance  of  disease,  685 
Non-symbiotic  nitrogen  fixation,  aerobic 
species,  402 

anaerobic  species,  402 

energy  relations,  404 


INDEX 


1027 


Non-symbiotic  nitrogen  fixation,  history 

of,  401 

Nosema  apis,  937,  942 
morphology,  940 

bombycis,  899 

morphology  of,  938 
in  pebrine,  937 

disease  of  bees,  939 

pulicis,  946 

Notonecta  glauca,  919 
Noxious  retention  theory  of  immunity, 

721 

Nuclein,  16 
Nucleolus,  1 6 
Nucleoplasm,  15 
Nucleus,  of  bacteria,  89 

diffuse,  17,  93 

division  of,  28 

forms,  17 

of  molds,  42 

of  protozoa,  125 

reproductive,  18 

structure  of,  15 

value  of,  1 6 

vegetative,  18 
Number  of  bacteria,  in  air,  306 

in  butter,  476 

in  concentrated  milk,  506 

in  cream,  476 

in  frozen  milk,  274 

in  ice  cream,  513 

in  infection,  674 

in  milk,  429 

in  soil,  367 

in  water,  316 

Nutrition  of  microorganisms,  195 
Nutrition,  energy  requirements,  199 

of  protozoa,  197 

OATS,  blade  blight  of,  956 
Obligate  aerobes,  228 

anaerobes,  228 

parasites,  132,  660 

saprophytes,  660 
Odors  in  milk,  428 
(Edema,  malignant,  839 


Oidium  albicans,  in  mouth,  593 
in  thrush,  776 

lactis,  58,  238,  272,  463 
in  butter,  482 
in  cheese,  503 
effect  of  pressure  on,  283 
symbiosis  of,  298 
Oleomargarine,  cultures  for,  482 
Olive  canning,  573 

knot,  969 

pickling,  572 
Oomycetes,  38 
Oospore,  38 

Ophidomonas  jenensis,  105 
Opsonic  index,  712 
Opsonins,  711 

autohemo-,  714 

hemo-,  714 

isohemo-,  714 
Opsonogens,  712 
Optimum  temperature,  269 
Orchids,  brown  rot  of,  982 
Organellae,  126 
Organic  acids,  as  food,  223,  226 

in  soil,  379 

Organic  food  preservatives,  556 
Organic  matter,  mineralization  of,  351 

in  soil,  358,  375 
Origin  of  soil,  41 7 
Ornothodoras  moubata,  901 
Oroya  fever,  897 
Osmosis,  174 

osmotic  pressure,  174,  176,  263 
Osteomyelitis,  792 
Outlines  of  plant  groups,  13 

of  protozoal  groups,  13 
Oxidase,  208,  216 
Oxidation  of  acetic  acid,  238 

of  acids,  238 

of  alcohol,  238 
•  of  ammonia,  201 

of  carbohydrates,  234 

of  carbon  monoxide,  377 
in  soil,  351,  373 

of  cellulose,  362 

of  hydrogen,  351,  377 


1028 


INDEX 


Oxidation  of  hyposulphite,  201 

influence  on  organisms  in  water,  320 

of  methane,  377 

of  minerals,  244 

of  nitrogen,  351,  374 

of  sulphur,  335 

in  water,  320 

Oxidizing  enzymes,  208,  216 
Oxygen,  as  food,  223,  226 

relations,  225 
Ozone,  in  purification  of  water,  325 

PACHYTYLUS  migratoroides,  917 
Parachrome  bacteria,  246 
Paramecium,  carbohydrate  in,  190 

caudatum,  127 

Parasites,  animal  in  food,  583 
facultative,  132,  660 
obligate,  132,  660 
of  uncertain  position,  139,  900 
Parasitic  microorganisms,  132,  196,  660 

molds,  48 

Parasitism,  132,  300 
Parenchymatous  tissues,  action  of  micro- 
organisms on,  682 
Parthenogenesis,  131 
Particles,  comparison  of  size  of,  182 
Pasteurellose,  avian,  808 
Pasteurization,  of  beer,  530 
of  condensed  milk,  531 
of  cream,  530 
of  foods,  530 
of  fruit  juices,  530 
of  milk,  459,  530 
Pathogenic  bacteria,  660 
in  butter,  485 
distribution  of,  662 
effect  of,  on  antibody  formation,  683 
on  blood-forming  organs,  68 1 
on  endothelial  tissues,  682 
on  epithelial  tissues,  682 
on  erythrocytes,  682 
on  leucocytes,  682 
on  metabolism,  68 1 
on  parenchymatous  tissues,  682 
in  food,  582 


Pathogenic  bacteria  of  intestine,  600 

in  milk,  452 

in  mouth,  595 

in  sewage,  338 
Pathogenic  protozoa,  660 
Peach,  leaf  spot  of,  975 
Pear  blight,  958 
Peas,  blight  of  stem,  956 

sterilization  of,  534 
Pebrine,  937 
Pellagra,  591,  865 
Pelomyxa  palustris,  15 

cellular  division  in,  32 

energids  of,  16 

mesomitosis  in,  32,  33 
Pemmican,  523 
"Penicillium,  234 

brevicaule,  53,  245 

camemberti,  53,  503 
enzymes  of,  215,  220 

characters,  51 

cultural  considerations,  52 

digitatum,  nitrogen  fixation  of,  362 

expansum,  52,  53,  654 
nitrogen  fixation  by,  362 

glaucum,  43 

temperatures  for,  270 

roqueforti,  53 

in  cheese,  502 
Pepsin,  204,  208,  212,  214 
Peptase,  208 
Peptones,  as  food,  223 

transformation  of,  398 
Percentage  index,  713 
Perfringens  antitoxin,  745 
Period  of  incubation,  680 
Periplaneta  americana,  907 
Perithecium,  39 
Permeability,  175 
Peroxidase,  216 
Perry,  628 

Pestularia  vesiculosa,  46 
Phagocytic,  index,  712 

theory  of  immunity,  723 
Phagocytosis,  711 
Phosphine,  242 


INDEX 


I02Q 


Phosphorus,  cycle  of,  262 

in  soil,  420 

Photogenic  bacteria,  251 
Phototaxis,  280 
Phototropism,  280 
Phycomycetes,  38,  61 

cell  structure,  40 

reproduction  of,  38 

Physical  changes,  in  canned  foods,  526 
Physical  forces,  adsorption,  171 

in  biological  activities,  155 

brownian  motion,  172 

colloids,  177 

crystalloids,  177 

dialysis,  175 

diffusion,  173 

dissociation,  159 

electrical  conductivity,  158 

energy,  155 

ionization,  159 

osmosis,  174 

permeability,  175 

plasmolysis,  177 

reaction,  160 

solutions,  156 

surface  tension,  168 
Physical  influences,  263 

electricity,  282 

light,  278 

mechanical  effects,  283 

temperature,  269 

water,  263 

Physiological  variation,  253 
Physiology  of  microorganisms,  145 
Pickles,  571 
Pieris  brassies,  945 
Pigments,  246 
Piroplasma  bigeminum,  661 

pathogenic,  894 
Plagiomonas,  135 
Plague,  830 

of  cattle,  856 

of  fowl,  860 
Plant  food  in  soil,  354 
Plants,  diseases  of,  949 

groups  of,  13 


Plasmodiophora  brassicae,  969,  971 
Plasmodium,  139 

cycle  of  development,  890 

falciparum,  in  malaria,  890,  892 

malariae,  661 
in  malaria,  890,  892 

parasitic,  890 

vivax,  in  malaria,  890,  892 
Plasmojysis,  177,  264 

changes  due  to,  89 
Plastids,  20 

Plating  methods  of  milk  analysis,  471 
Pleuro-pneumonia,  bovine,  856 
Plum,  leaf  spot  of,  975 
Pneumococcus,  799 
Pneumomycosis,  775 
Pneumonia,  antiserum  for,  746 

bovine  pleuro-,  856 

broncho-,  795 

lobar,  798 

vaccine,  738 
Poisoning,  beriberi  from,  591 

botulism  from  food,  587 

by  cheese,  586 

chemical  nature  of,  592 

classes  of  food,  582 

due  to  saprophytic  bacteria,  584 

ergotism  from  food,  591 

by  fish,  585 

by  foods,  581 

by  ice  cream,  586 

infections  transmitted,  582 

kakke  from  food,  591 

by  meat,  584 

by  milk,  586 

pellagra  from  food,  591 

by  sausage,  584 

by  shell  fish,  586 

by  vegetables,  587 
Poisons,  288 

Polar  bodies,  of  protozoa,  130 
Poliomyelitis,  epidemic,  864 

control  in  London,  Canada,  763 
Pombe,  631 
Porter,  622 
Porthesia  chrysorrhea,  919 


1030 


INDEX 


Porthetria  dispar,  909,  910,  935,  947 
Potassium  in  soil,  424 
Potato,  blackleg  of,  980 

wilt  of,  990 
Potential,  159 

Poultry,  refrigeration  of,  544 
Powdered  milk,  507 
Precipitate,  718,  720 
Precipitin,  718 

anti-,  719 

auto-,  719 

co-,  720 

forensic  use  of,  720 

formation  of,  718 

iso-,  719 

normal,  697,  718 

specific  inhibition,  719 
Precipitinogen,  718,  720 
Precipitoid,  719,  720 
Predisposition  to  disease,  685 
Preservation  of  food  by  chemicals,  550 
legal  control  of,  558 

by  cold,  542 

by  drying,  516 

by  heat,  524 

by -pasteurization,  530 

of  vegetables  by  fermentation,  570 
Preservatives,  288 

acetic  food,  556 

alcohol,  557 

benzoic  acid,  556 

boric  acid,  555 

effect  on  food,  554 

formaldehyde,  557 

formic  acid,  556 

inorganic,  555 

in  milk,  460 

nitric  acid,  555 

nitrous  acid,  555 

organic,  556 

ozone,  325 

salicylic  acid,  556 

sodium  chloride,  554 

sugar,  554 

sulphurous  acid,  555 

wood  smoke,  557 


Pressure,  influence  of,  283 
Proagglutinoids,  717 
Processing  of  canned  foods,  532 
Prodigiosin  bodies,  247 
Production  of  heat,  250 

of  light,  251 

Prohibition  and  wine,  621 
Promitosis,  of  protozoa,  130 
Properties  of  enzymes,  205 
Prophase,  28 

Propionic  bacteria,  in  wine,  610 
Prorodon  teres,  26 
Protease,  208,  211 
Protection  against  disease,  688 

conjunctiva,  692 

cutaneous  orifices,  688 

exposed  mucous  membranes,  689 

geni to-urinary  tract,  692 

inflammatory  processes,  693 

intestines,  691 

lungs,  691 

mouth,  690 

nasal  cavity,  690 

natural    antibacterial    substances, 

695 

natural  antitoxins,  694 

normal  agglutinins,  696 
hemolysins,  696 
precipitins,  697 

skin,  688 

stomach,  691 

subcutaneous  tissue,  689 
Protein  bodies,  240 

in  soil,  380 
Protein,  in  cells,  188 

determination  of  degradation,  240 

enzymes  of,  208,  211 

foods,  drying  of,  523 

products  of  degradation,  239 

as  reserve  product,  22 

in  sewage,  332 

toxic  bacterial,  676 
Proteolysis  in  cheese,  494 
Proteolytic  enzymes,  208,  211 

determination  of,  212 

of  yeasts,  215 


INDEX 


1031 


Proteosoma,  139 
Proteus  alveicola,  946 
Proteus  group,  312 
Protista,  n,  12,  117 

metachromatic  corpuscles  of,  44 
nucleus  in,  91 
Protomitosis,  31 
Prototrophic  organisms,  223 
Protozoa,  activities  of,  126 
locomotion,  127 
cilia,  127 
flagella,  127 
myonemes,  127 
pseudopodia,  127 
undulating  membrane,  127 
chromidia  in,  18 
classification  of,  133 
cultivation  of,  142 
diseases  caused  by,  876 
Protozoa,  functions  of,  1 26 
general,  123 
groups,  13,  14 
metabolism  of,  198 
nutrition  of,  197 
parasitism,  132 
commensals,  133 
facultative  parasites,  132,  660 
obligatory  parasites,  132,  660 
saprozoic  parasites,  132 
pathogenic,  660 
reproduction  of,  128 
anisogamous,  131 
autogamy,  131 
binary  division,  128 
chromatic  reduction,  130 
chromosomes,  130 
conjugation,  131 
copula,  131 
copulation,  130 
encystment,  132 
gametocytes,  131 
gemmulation,  128 
isogamous,  131 
life  cycle,  131 
macrogametes,  131 
merozoites,  129 


Protozoa,  reproduction  of,  mesomitosis, 
130 

microgametes,  131 

mitosis,  130 

parthenogenesis,  131 

polar  bodies,  130 

promitosis,  130 

schizogony,  129 

self-fertilization,  131 

sporogony,  129 

sporozoites,  129 

zygote,  131 
in  soil,  364 
structure,  124 

chromatin,  125 

chromidia,  126 

cytoplasm,  125 

ectoplasm,  125 

endoplasm,  125 

linin,  126 

nucleus,  125 

organellae,  126 
technique  for  study  of,  140 
in  water,  320 
Protoxoids,  704 

Pseudograsserie  of  gipsy  moth  cater- 
pillar, 930 
Pseudomonas  aptatum,  976 

morphology  of,  977 
avenae,  956 
beticola,  972 

morphology,  972 
campestris,  978 

morphology,  978-979 
citri,  973 

morphology,  973 
fluorescens,  in  soil,  369 
hyacinthi,  979 

morphology  of,  980 
juglandis,  963 

morphology  of,  965 
lachrymans,  974 

morphology  of,  974 
medicaginis,  951 

morphology  of,  953 
mori,  955 


1032 


INDEX 


Pseudomonas  mori,  morphology  of,  955 
phaseoli,  953 

morphology  of,  954 
pisi,  956 

morphology  of,  957 
pruni,  975 

morphology  of,  976 
pyocyanea,  403,  726 

agglutinins  in  blood,  697 

antibiosis  of,  300 

antitoxin  for,  704    * 

flagella,  105 

in  fowl  diphtheria,  818 

hemolysis  of,  682 

pathogenic  in  intestine,  600 

pigment  of,  246 

in  protein  degradation,  243 

proteolytic  enzyme  of,  213 

toxin  of,  676 

radicicola,  403,  407,  408,  409,  412, 
420 

prototrophic,  223 

resistance  to  drying,  268 

symbiosis  of,  298 

temperature  for  growth,  354 
solanacearum,  990 

morphology,  991 
steward,  989 

morphology,  990 
syncyanea,  flagella  of,  105 
tumefaciens,  966 

morphology,  968 
viridilividum,  954 

morphology,  954 
Pseudopodia,  24,  127 
Pseudo-yeasts,  75 

on  grapes,  606 
Ptomains,  in  food  poisoning,  592 

production  of,  241 
Ptyalin,  208 

Puerperal  septicaemia,  795 
Pulsating  vacuoles,  22 
Purification  of  water,  by  chemicals,  326 
nitration,  323,  324,  325 
heat,  326 
ozone,  325 


Putrescin,  241 
Putrid  cheese,  499 
Pyaemia,  792 
Pyocyanase,  300 
Pyorrhoea  alveolaris,  595 
Pyrameis  cardui,  946 
Pyrinin,  16 

RABBITS,  coccidiosis  of,  889 
Rabies,  868 

control  of,  in  London,  Canada,  763 

serum  for,  746 

vaccine,  731 
Radium  rays,  281 
Ragi,  635 

Rain,  microorganisms  in,  316 
Ranatra  linearis,  919 
Rate  of  oxidation,  351,  373 

carbon  in  soil,  351,  373 

hydrogen,  351 

nitrogen  in  soil,  351,  374 
Reaction,  160 

of  pathogenic  organisms  on  body, 
680 

of  soils,  355 

transformation,  in  soils,  372 
Receptacles  for  canning,  536 
Reducing  enzymes,  208,  216 
Reductase,  208,  216 
Reduction  of  nitrates,  335 

nitrites,  337 

sulphate,  335 
Red  water,  894 
Refrigeration,  542 

after-storage  changes,  544 

of  butter,  547 

chilling  changes,  543 

effect  upon  food,  542 

of  eggs,  547 

of  fish,  544 

of  fruits,  548 

legal  control  of,  549 

of  meat,  544 

of  milk,  547 

of  poultry,  544 

storage  changes,  543 


INDEX 


1033 


Refrigeration  of  vegetables,  548 
Refuse,  disposal  of  canning  factory,  540 
Relapsing  fever,  901 
Relationship  of  bacteria,  117 
Rennet,  205,  208,  213 

proteolytic  action  of,  495 
Reporting  of  births,  761 

of  deaths,  761 

of  infectious  diseases,  761 
Reproduction,  bacteria,  83 

cell,  27 

molds,  43 

protozoa,  128 

spirochaetes,  107 

trichobacteria,  108 

yeasts,  61 
Reserve  products,  22 

of  bacteria,  101 

of  molds,  44 
Resistance  to  infection,  675 

of  spores,  276 

Retention  theory  of  immunity,  721 
Retting,  658 

Reversibility  of  enzymic  action,  218 
Rhinosporidium  kinealyi,  139,  898 
Rhizobium  beyerincki,  410 

radicicola,  410 
Rhizopoda,  characteristics,  134 

diseases  caused  by,  876 
Rhizopus,  fermentation,  50 

javanicus,  51 

nigricans,  43,  49 
fruiting  stolons  of,  49 
phototropism  of,  279 

oryzae,  51 

enzymes  of,  220 
Rhyzotrogus  solsticialis,  946 
Rice  beer,  630 
Ringworm,  777 
Ripening  of  cream,  477 
Rivers,  microorganisms  in,  318 
Rivularia  bullata,  nucleus,  17 

threads,  17 
Romalea  miles,  946 
Ropy  milk,  464 
Roquefort  cheese,  502 


Rot,  of  cabbage,  978 

of  callajily,  984 

of  carrot,  984 

of  cauliflower,  983 

of  cocoanut,  981 

of  hyacinth,  985 

of  muskmelon,  986 

of  orchids,  982 

of  potato,  980 

of  sugar  beet,  987 
Rotation  of  elements,  258 
Routes  of  infection,  669,  754 
Rum,  631 

Jamaica,  632 

SACBROOD,  of  bees,  931 
Saccharomyces,  61 

albicans,  in  thrush,  776 
anomalus,  62 

in  beer,  627 

spores  of,  63 

in  wine,  609 
apiculatus,  77 

in  beer,  627 

on  grapes,  607 

in  wine,  75,  614,  615 
cerevisiae,  65,  74 

in  beer,  622 

in  bread,  564 

in  brewing,  73 

cells  of,  1 6 

in  compressed  yeast,  563 

cytology  of,  64 

effect  of  pressure  on,  283 

occurrence,  61 

oxidation  of,  226 

spore  formation  in,  27 

temperatures,  270 

variability  in,  253 

in  white  lead,  654 
ellipsoideus,  66,  74 

in  beer,  625,  627 

in  compressed  yeast,  563 

on  grapes,  606,  607 

occurrence,  61 

in  white  lead  production,  654 


1034 


INDEX 


Saccharomyces,    ellipsoideus    in    wine, 

73,  614,  615 
exiguus,  in  beer,  627 
farciminosus,  morphology,  783 
in  mycotic  lymphangitis,  782 
fcetidus,  in  beer,  627 
fragilis,  74 
kefir,  510 

ludwigii,  63,  71,  72 
sporulation  in,  70 
marxianus,  62 

pasteurianus,  in  beer,  625,  627 
in  brewing,  75 
on  grapes,  606 
spores  of,  63 
temperatures,  270 
thermal  deathpoint,  276 
pyriformis,  74 

in  ginger  beer,  631 
(schizo)  octosporus,  63,  68 
conjugation  in,  34 
cytological  phenomena  in,  70 
(schizo-)  pombe,  69,  75,  77 
vordermanni,  74 

in  alcohol  production,  635 
zopfi,  in  sugar,  580 ' 
Sake,  Japanese,  630 

aspergillus  in  preparation  of,  56 
Salicylic  acid,  as  preservative,  556 
Sand  filter,  323,  341 

life  in,  339 
Saprolegniaceae,  60 
Saprophytes,  cosmopolitan,  47 
facultative,  660 
obligate,  660 
Saprophytic  organisms,  196 

food  poisoning  from,  584 
Saprozoic  organisms,  196 

parasites,  132 
Sarcina,  87 

aurantiaca,  in  air,  307 
Iutea,3i2,  387,  388 

in  air,  307 
ventricula,  596 

Sarcocystis  tenella,  metachromatic  cor- 
puscles of,  23 


Sarco-peptone,  523 
Sarcosporidia,  124,  139 

parasitic,  898 
Sauerkraut,  570 
Sausage  poisoning,  584 
Scarlet  fever,  855 
Schick  test,  753 
Schistocerca  americana,  917 

pallens,  912 

paranensis,  917 
Schizogony,  129 
Schizosaccharomyces,  62 

octosporus,  63,  68 
conjugation  in,  34 
cytological  phenomena  in,  70 

pombe,  69,  75,  77 
Sclerotinia  fuckeliana,  605 
Sclerotium,  37 
Scopulariopsis  repens,  53 
Sea  water,  microorganisms  in,  318 
Sedimentation,  influence  on  germ  con- 
tent of  water,  320,  322,  323 
Selected  milk,  432 
Selenopsis  gemminata,  918 
Self-fertilisation,  131 
Sensitized  vaccine,  739 
Septic  sore  throat,  from  food,  583 
Septic  tank,  342 
Septicaemia,  of  caterpillar,  918 

of  cockchafer,  925 

general,  795 

hemorrhagic,  825 

of  larva  of  Lamellicornac,  929 

puerperal,  795 
Septum,  36 
Serum,  antidiphtheritic,  741 

antidysenteric,  747 

anti-gas  gangrene,  745 

antigonococcic,  746 

anti-hog  cholera,  746 

antimeningococcic,  745 

antimicrobial,  740 

antiperfringens,  745 

antipneumococcic,  746 

antirabic,  746 

anttetreptococcic,  745 


INDEX 


1035 


Serum,  antitetanic,  744 
antitoxic,  740 
Dorset-Niles,  746 
hyperimmune,  746 
preservation  of,  747 
Sewage,  anaerobic  organisms,  332 
disinfection,  343 
fermentation  of,  332 
cellulose,  333 
fats,  334 
proteins,  332 
urea,  335 
filters,  341 
longevity  of  pathogenic  organisms 

in,  338 

microorganisms  of,  330,  331 
cultivation  of,  340 
destruction    by    biological    proc- 
esses, 343 

by  chemical  processes,  343 
life  in  filters  of,  339 
oxidizing  bacteria,  337 
pathogenic  bacteria  in,  338 
prevalence  of  pathogenic,  338 
reduction  of  nitrates,  335 

sulphates,  335 
septic  tank  for,  342 
streptococci  in  water,  313,  330 
tanks,  342 
Sexual  changes,  34 
Sheath  of  higher  bacteria,  108 
spirochaetes,  108 
trichobacteria,  109 
Sheeppox,  857 
Shell-fish  poisoning,  586 
Shoyu  preparation,  aspergillus  in,  56 
Side  chain  theory  of  immunity,  722 
Signatera,  585 
Silage,  574 

B.  botulinus  in,  576 
fermentation  of,  575 
Silkworm,  B.  sotto  in,  945 

pebrine  of,  937 
Skin,  protection  by,  688 
Sleeping  sickness,  882 
Slimy  milk,  464 


Slimy  wine,  610 
Smallpox,  854 

vaccine,  727 

Snow,  microorganisms  in,  317 
Sodium  chloride  as  preservative,  554 
Soil,  acidity,  355 

causes  of,  355 

change  in,  produced  by  organ- 
isms, 357 

number     of     organisms,     influ- 
enced by,  357 
range  of,  355 

species,  influenced  by,  357 
aeration,  350 
aerobic  activities,  350 
algae  in,  364 
aluminum  in,  425 
ammonia   formation,    387 

transformation,  398 
ammonification,  383 

climatic    conditions    influencing, 

384 
efficiency,  388 

of  different  species,  387 
experimental  study,  383 
mechanism  of,  384 
molds,  361 

numbers  involved,  385 
species  involved,  385 
anaerobic  activities,  350 
antagonism  in,  426 
bacteria,  367 

aerobic  species,  402 
anaerobic  species,  402 
counting,  methods  of,  370 
at  different  depths,  368 
distribution,  368 
groups(morphological  and  physio  • 

logical),  369,  370 
methods  of  study  (quantitative 

and  qualitative),  371,  372 
nitric,  390 
nitrous,  390 
number,  367 
productive  soil,  367 
sulphur,  422 


1036 


INDEX 


Soil,  bacteria,  unproductive  soil,  367 

in  water,  312 
bacterial  substance  available,  398 

substance  present,  397 
biological    factors    of    soil-forma- 
tion, 417 

calcium,  374,  417 
carbohydrates  (origin),  375 
carbon  dioxide  in,  351 
carbon-nitrogen  ratio,  382 
climatic  influences,  352 
colloidal  nature  of,  349 
composition  (mechanical),  250 
copper,  425 
culture  medium,  346 
cultures  for  soil,  414 
denitrification,  395 

environmental  relations,  396 

experimental  study,  395 
drought,  348 
early,  353 
fermentations,  cellulose,  362,  375 

fats,  378 

fixation  of  nitrogen,  400 

organic  matter,  345 

proteins,  380 

starches,  378 

sugars,  378 

waxes  and  gums,  378 
food  (plant)  assimilation  of,  354 

(plant)  production  in,  354 
formation  of,  417 
general  discussion,  345 
higher  plants  in,  367 
hydrogen  ion  concentration,  356 
infection,  666 
inoculation,  412 
iron,  425 
late,  353 

magnesium,  374,  417,  419 
manganese,  425 
medium,  as  culture,  290 
mineral  food,  358 
mineralization  of    organic    matter, 

3Si 
moisture  relations,  346 


Soil,  moisture,  excessive,  348 

rainfall,  346 

range  of,  347 
molds  in,  360 

nitrates,  transformation  of,  398 
nitrification,  389 

bacteria     (nitric    and    nitrous), 

390 

environmental  relations,  391 
experimental  study,  389 
nitrates,  accumulation,  394 
disappearance,  394 

nitrogen,  addition  of,  374 
utilization,  molds,  362 

nitrogen     (atmospheric)     fixation, 

aerobic  species,  362,  402 
anaerobic  species,  402 
biological  relations,  401 
chemical  relations,  401 
cultures  for  inoculation,  414 
development  of  organisms,  408 
energy  relations,  404 
entrance  of  organisms,  408 
environmental  relations,  410 
inoculations,    methods    of,    412, 

414 

mechanism,  410 
physiological  efficiency,  409 
resistance  of  plants  to,  409 
specialization  of  organisms,  410 
symbiotic,  history  of,  405 
symbiotic  (non),  history  of,  401 
theories  of,  400 
variations  of,  410 

nitrogenous   compounds,    transfor- 
mation of,  398 

organic  acids,  accumulation  of,  380 
source  of,  379 
transformation  of,  380 

organic  matter  in,  358 

origin  of,  417 

carbohydrates  in,  375 
fats,  378 
waxes,  378 

oxidation  of  carbon,  351,  373 
carbon  monoxide,  377 


INDEX 


Soil,  oxidation  of  hydrogen,  351,  377 

methane,  377 

nitrogen,  351,  374 
peptone,  transformation  of,  398 
phosphorus,  374,  420 
plants  (higher  in),  367 
potassium,  424 
production  of  hydrogen,  375 

methane,  375 
protein  bodies  in,  380 

amount,  380 

quality,  380 
protozoa  in,  364 
reaction  of,  355 
reduction  of  nitrates,  394 

sulphates,  423 

seasons,  influence  of,  352,  368 
sterilization  of,  366 
sulphate  reduction,  423 
sulphofication,  423 
sulphur,  374,  421 
temperature  relations,  352 
temperatures  of,  352 
transformation    of    nitrogen    com- 
pounds, 383,  398 

organic  acids,  380 

reactions  of,  355,  372 
weatheiing  process,  417 
Soluble  enzymes,  214 

toxins,  676 
Solutions,  156 

alkalinity  of,  161 

colloidal.  157,  179 

crystalloidal,  158 

diphasic,  157 

disperse  phase  of,  157,  179 

dispersion  means  of,  157,  179 

dispersoids,  157,  179 

electrolytic,  158 

emulsoid,  157,  179 

heterogeneous,  157 

homogenous,  157 

hydrogen  ion  concentration  of,  162 

influence  of,  265 

neutrality  of,  161 

non-electrolytic,  158 


Solutions,  one-phase  system,  157 

polyphase,  157 

reaction  of,  160 

solute,  157 

solvent,  157 

suspensoid,  157,  179 

true,  156 
Solvent,  157 
Somatose,  523 
Sources  of  organic  acids  in  soil,  379 

carbon  for  microorganisms,  222 

hydrogen  for  microorganisms,  223 

infection,  664 

microorganisms  in  milk,  434 

minerals  for  microorganisms,  224 

nitrogen  for  microorganisms,  223 

oxygen  for  microorganisms,  223 
Special  milk,  432 
Specific  inhibition,  719 
Spindle,  achromatic,  28 
Spireme,  28 
Spirilliaceae,  107 
Spirillum,  desulphuricans,  424 
in  sewage,  336 

rubrum,  flagella,  25,  105 

rugula,  in  sewage,  334 

sputigenum,  594 

undula,  105 

volutans,  cilia  of,  107 

metachromatic  corpuscles  of,  101, 

102 
Spirochaeta,  anserina,  904 

buccalis,  594,  900 

dentium,  900 

duttoni,  morphology,  902 

gallinarum,  904 

macrodentium,  594,  595 

media,  594 

microdentium,  594 

pallida,  in  mouth,  595 
in  luetin  preparation,  753 

recurrentis,  in  relapsing  fever,  900, 
901 

vincenti,  900 
Spirochaetes,  107 

cell  aggregates,  108 


io38 


INDEX 


Spirochaetes,  characteristics,  139 

diseases  caused  by,  900,  904 

form  of,  107 

motility,  107 

parasitic,  900 

as  protozoa,  118 

reproduction  of,  107 

sheaths  of,  108    • 

size  of,  107 

Spirophyllum  ferrugineum,  no 
Spoilage  of  canned  foods,  540 
Spongomonas  uvella,  26 
Sporangiophore,  50 
Spores,  of  bacteria,  arrangement,  85 
germination  of,  86 
resistance,  85 

resistance  to  heat,  276 

in  canning,  536 
Sporogony,  27 

in  protozoa,  129 
Sporotrichum  globuliferum,  944 
Sporozoa,  137 

diseases  caused  by,  888 
Sporozoites,  129 
Sporulation,  of  ascospores,  67 
Spot,  angular  of  cucumbers,  974 

of  larkspur,  975 

of  nasturtium  leaves,  976 

of  plum  and  peach,  975 

of  sugar  beet,  976 
Standards  of  milk,  471 

for  water,  321 

Staphylococcic  infections,  792 
Staphylococcus,  87 

pyogenes  aureus,  784 
in  pneumonia,  798 
Starch  fermentation,  237 

production  of,  579 
Starters,  for  beer,  625 

butter,  477,  481 

buttermilk,  512 

cheese,  488 

milk  drinks,  507 

vinegar,  641 

wine,  614 
Stauronautus  maroccanus,  915,  917 


Steapsin,  208,  211 
Sterigma,  39 
Sterigmatocystis,  54 

nigra,  654 

Sterilization,  agitation  of  container  dur- 
ing, 538 

consistency  of  material  for,  538 
of  corn,  534 

economic  considerations,  532 
of  fish,  534 
of  fruits,  535 

initial  temperature  of,  538 
of  meat,  533 
of  peas,  534 

size  of  container,  relation  of,  538 
of  soil,  365 

spores,  relation  of,  536 
Stilton  cheese,  502 
Stolon,  50 
Stomach,  microorganisms  of,  596 

protection  by,  691 
Stomoxys,  887 
Straining  of  milk,  453 
Streak  disease  of  sweet  peas  and  clover, 

962 

Streptobacilli,  88 
Streptococci  in  sewage,  313,  330 
Streptococcic  infections,  795 
Streptococcus,  87 

apis,    in    European    foul    brood, 

928 
bombycis,  in  flacherie,  907 

morphology  of,  908 
disparis,  909 

morphology  of,  910 
faecalis,  797 
haemolyticus,  795 
in  mouth,  594 

in  production  of  vaccine,  738 
lacticus,  235,  237,  446 

absence  of  katalase  in,  216 
inhibition  of  growth,  301 
mesenteroides,  capsule,  104 

in  sugar,  580 

morphology   and    cultural    charac- 
teristics, 796 


INDEX 


1039 


Streptococcus,  pneumoniae,  in  conjunc- 
tiva, 692 

cultural  characteristics,  799 
in  healthy  persons,  663 
in  lungs,  691 
in  pneumonia,  798 
for  serum  production,  746 
in  tonsils,  670 
vaccine,  737 

variation  in  infection,  673 
pyogenes,  445,  662 

for  antiserum  production,  745 
antitoxin  for,  704 
dissemination  of,  677 
elimination  of,  679 
in  genital  tract,  672 
in  healthy  individuals,  663,  688 
hemolysins  of,  682 
in  infections,  795 
in  nasal  cavity,  690 
in  pneumonia,  798 
in  tonsils,  670 
toxin  of,  676     - 
vaccine  for,  737 
variation  in  infection,  673 
virulence,  674 
salivarius,  in  mouth,  594 
viridans,  795 

in  mouth,  594,  595 
Stump  root  of  cabbage,  969 
Subcutaneous  tissue,  protection  by,  689 
Subsidence  in  water,  319 
Sucrase,  210 
Sugar,  in  fermentation,  233 

production  of,  580 
Sugar  beet,  soft  rot  of,  987 
leaf  spot  of,  976 
tuberculosis  of,  972 
Sulphate  reduction,  423 
in  sewage,  335 
in  soil,  423 
Sulpho-bacteria,  93,  105,  107,  in 

in  soil,  422 
Sulphofication,  423 
Sulphur  cycle,  262 
oxidation,  237 


Sulphur  in  soils,  421 

Sulphurous  acid  as  preservative,  555 

Surface  tension,  168,  169 

film,  169 

molecular  pressure,  169 
Surface  washings  in  water,  312 
Surra,  887 
Susceptibility,  684 

definition,  684 

familial,  688 

general,  684 

hyper,  684 

individual,  688 

natural,  687 

racial,  687 

Suspensions  for  agglutination  tests,  751 
Swamp  fever,  873 
Sweet  corn,  wilt  of,  989 

curdling  of  milk,  464 
Swine  erysipelas,  832 
Swiss  cheese,  50x5 
Symbiosis,  297 
Symbiotic  nitrogen  fixation,  cultures  in, 

4U 

development  of  organisms,  408 

entrance  of  organisms,  408 

environmental  relations,  410 

history  of,  405 

inoculation,  methods  of,  412,  414 

legume-earth  for  inoculation,  414 

mechanism  of,  410 

physiological  efficiency,  409 

resistance  of  plants,  409 

specialization,  410 

variations,  410 
Symptomatic  anthrax,  839 

vaccine,  729 
Syphilis,  903 

test  for,  708 

test  by  luetin,  753 
Syrups,  preservation  of,  522 

TAFFIA,  632 
Taints  in  milk,  428 
Tanks,  anaerobic,  342 
septic,  342 


1040 


INDEX 


Tanning,  655 
Tapej,  635 
Tea,  580 
Telophase,  "29 
Temperance  beer,  622 
Temperature,    cardinal    points,    signifi- 
cance of,  272 

endpoints  of  fermentation,  273 

freezing,  274 

influence  on  growth,  269 
on  germ  content  of  milk,  458 
of  water,  319 

initial  for  canning,  538 

maximum,  271 

minimum,  270 

optimum,  269 

resistance  of  spores,  276 

of  silage  fermentation,  575 

of  soil,  352 

thermal  death-point,  275 

of  wine  fermentation,  617 
Tests  for  milk  quality,  489 
Tetanus,  842 

antitoxin,  744,  845 

toxin,  744 
Tetracoccus,  87 
Texas  fever,  894 
Thamnidium  elegans,  41,  51 

general  characters,  51 
Theories  of  cheese  ripening,  492 

of  immunity,  721 

of  nitrogen-fixation,  400 
Thermal  death-point,  275 
Thermophilic  bacteria,  271 

in  canned  foods,  529 
Thiobacillus  denitrificans,  396,  423 
Thiobacteria,  107 
Thiosulphates,  as  food,  224 
Thiothrix  nivea,  in 

tenuis,  17 

Thrombase,  208,  213 
Thrush,  776 
Tobacco,  578 

wilt  of,  990 
Tomato  blight,  963 

wilt  of,  990 


Tortilla,  Mexican,  568 
Torula,  61,  77 

in  beer,  75,  622 

on  grapes,  608 

in  salt  solution,  265 
Toxic  proteins,  676 
Toxin-antitoxin  mixture,  739 
Toxins,  248,  740 

cyto-,  705,  709 

diphtheria,  741 

endo-,  676 

leucocyte-,  710 

neutralization,  702 

nori-microbial,  249 

soluble,  676 

tetanus,  744 

true,  704 
Toxoid,  true,  704 
Toxon,  704 

Toxophile  receptors,  700 
Toxophore  group,  700 
Toxoplasma,  900 
Transformation  of  ammonia,  398 

nitrates,  398 

nitrogen  in  soil,  383 

organic  acids  in  soil,  380 

peptone,  398 

reactions  in  soil,  372 
Treponema  pallidum,  121,  66 1,  708 
in  genital  tract,  671,  692 
germinal  infection  with,  672 
morphology,  903 

parasitic,  903 

pertenue,  in  yaws,  904 
Trichobacteria,  107,  108 

branching  of,  108 

reproduction  of,  108 

sheath,  109 
Trichocyst,  26 
Trichoderma  koeningi,  389 
Trichomonas,  characteristics,  36 

eberthi,  136 

intestinalis,  in  faeces,  599 

pathogenic  in  intestine,  600 
Trichomycosis,  777 
Trichophyton,  tonsurans,  777 


INDEX 


1041 


True  solutions,  156,  179 
Trypanoplasma,  characteristics,  135 
Trypanosoma,  135 

americanum,  888 

brucei,  661,  887 

cruzi,  morphology  of,  886 

dimorphon,  887 

diseases  caused  by,  88 1 

equinum,  887 

equiperdum,  888 

evansi,  887 

gambiense,  882 
morphology,  882 

granulosum,  882 

lewisi,  66 1,  887,  888 

percae,  135 

rhodesiense,  882 

rotatorium,  fat  globules  in,  23 

theileri,  888 

tincae,  135 
Trypanosomiases,  animal,  887 

human,  886 

treatment  of,  885 
Trypsin,  204,  208,  212,  214 
Tryptase,  208 
Tryptophan,  241 
Tubercles,  root,  408 
Tuberculin,  747,  835 

Koch's  old,  747 

Koch's  other  (T.  R.  and  B.  E.),  749 
Tuberculosis,  833 

of  animals,  833 

avian,  834 

control  of,  837 

in  London,  Canada,  763 

from  food,  583 

of  sugar  beet,  972 

tests  for,  749 

vaccine,  736 

Tyndall's  phenomenon,  181 
Types  of  cheese,  486 
Typhoid  fever,  314,  315,  845 

agglutination  test  for,  752 

control  of,  850 

transmitted  in  food,  584 

vaccine,  737 


Typhus  fever,  874 
Tyrosin,  241 
Tyrosinase,  208,  216 

UDDER  of  cow,  434 

bacteria    from   different   quarters, 

436 
from  vrhole  udder,  435 

diseased,  437 

healthy,  434 

interior,  434 

wiping,  effect  on  germ  content  of 

milk,  442 

Ultramicroscope,  181,  183 
Ultramicroscopic  organisms,  66 1 
Undulating  membrane,  25,  127 
Unit  of  biological  activity,  147 
Urea  bacteria,  enzyme  of,  215 
Urea  decomposition,  202,  335 

equation  of  fermentation,  225 

production  of,  243 
Urease,  208,  214 
Uric  acid,  production  of,  243 
Urospora  lagidis,  mesomitosis  of,  33 
Utensils,  relation  to  milk,  440,  444 

VACCINE,  712 

anthrax,  735 

Asiatic  cholera,  738 

bacterial,  736 

blackleg,  729 

bubonic  plague,  739 

canine  distemper,  738 

hog  cholera,  733 

influenza-pneumonia,  738 

manufacture  of,  724 

methods  of  preparation,  725 

pneumonia,  738 

rabies,  731 

sensitized,  739 

smallpox,  727 

tuberculosis,  736 

typhoid  fever,  737 
Vacuoles,  22 

pulsating,  20 
Vanessa  urticae,  931,  945 


1042 


INDEX 


Variation  in  infection,  672 

in  numbers  of  bacteria  in  soil,  368 

physiological,  253 
Vegetables,  chemical  preservation  of,  553 

food  poisoning  from,  587 

refrigeration  of,  548 

sterilization  of,  534 
Vegetation,    influence  on  organisms  in 

water,  320 
Venereal  disease,  control  of,  in  London, 

Canada,  764 
Vermicelli,  522 
Vinegar,  acetic  fermentation,  636 

after-treatment  of,  647 

apparatus  for  manufacture  of,  641 

bacteria,  636,  638 

cultures  for,  641 

diseases  of,  647 

domestic  method,  641 

fermentation  of,  640 

film,  function  of,  647 

manufacture  of,  639 

nature  of,  636 

Orleans  method,  642 

Pasteur  method,  643 

rapid  methods,  644 

raw  materials  of,  639 

rotating  barrel  method,  646 

starters  for,  641 
Vinegar-oxidase,  208,  214,  216 
Virulence  of  infection,  674 
Virus,  attenuation  of,  725 

filtrable,  66 1 

of  flacherie,  935 

of  sacbrood,  933 
Vitamines,  194 
Volutin,  45 

WALKER'S  hyacinth  disease,  979 
Walnut  blight,  963 
Wassermann  test,  708 
Water,  analysis  (qualitative  and  quan- 
titative), 321,  322 
B.  coli  in,  313 
enteritidis  in,  313 
typhosus  in,  314 


Water,  bacteria  of  natural,  311 
Bact.  lactis  aerogenes  in,  314 
-borne  infections,  666 
classes  of  bacteria  in,  311 
coagulating  basins,  324 
decrease  of  organisms  in,  319 
dilution  of,  320 
filters,  323,  325 
nitrations,  323,  324,  325 
food  influences,  319 
from  hail,  germ  content,  317 
increase  of  organisms  in,  319 
lake,  germ  content,  318 
light,  influence  of,  319 
microorganisms  in,  310,  311 
Microspira  comma  in,  314 
oxidation,  influence  on  germ  con- 
tent, 320 

protozoa,   influence   on  germ  con- 
tent, 320 

purification,  by  chemicals,  326 
by  heat,  326 
by  ozone,  325 

from  rain,  germ  content,  316 
from  rivers,  germ  content,  318 
sea  water,  germ  content,  318 
sedimentation  of,  320,  322,  323 
sewage  streptococci  in,  313 
from  snow,  germ  content,  317 
soil  bacteria  in,  312 
as  source  of  infection,  666 
supply  for  canning,  536 

for  dairy,  441 
surface  washings  in,  312 
temperature,  influence  in,  319 
in  upland  surface,  318 
vegetation,  influence  on  germ  con- 
tent, 320 
from  wells,  germ  content,  317,  326 

Wax  fermentation,  378 
in  soil,  378 

Weathering  process  in  soil,  417 

Weight  of  bacterial  cell,  221 

Weisbiers,  622 

Wells,  construction  of,  326,  328 
location  of,  326,  327 


INDEX 


1043 


Wells,  microorganisms  in,  317 

Whiskey,  631 

White  diarrhoea,  bacillary,  806 

White  lead,  production  of,  654 

Whooping  cough,  825 

Widal  test,  714 

Wilt,  of  cucurbits,  988 

of  eggplant,  990 

Irish  potato,  990 

sweet  corn,  989 

tobacco,  990 

tomato,  990 

Wilt  disease  of  gypsy  moth,  935 
Wine,  603 

acetic  bacteria  in,  609 

aerobic  organisms  in,  608 

anaerobic  organisms  in,  609 

butyric  bacteria  in,  612 

classification  of,  603 

composition  of,  604 

control  after  fermentation,  618 
before  fermentation,  612 
of  fermentation,  615 

defined,  603 

dry,  603 

fermentation,  615 

fining  of,  620 

fortified,  603,  604 

lactic  bacteria  in,  610 

mannitic  bacteria  in,  611 

as  medium  for  cultivation,  603 

microorganismsjn,  608 

mycodermae  in,  608 

and  prohibition;  621 

propionic  bacteria  in,  610 

red,  603 

slime-forming  bacteria  in,  610 

sparkling,  603 

starter  for,  614 

sweet,  603 

white,  603 

yeasts  of,  74 
Wisconsin  curd  test,  400 
Wood  smoke  as  preservative,  557 


Wort,  623 

production  of,  561 
Wounds,  792 

X-RAYS,  281 

YAHOURTH,  510 

Yaws,  904 

Yeast,  ash  elements  in,  193 

of  beer,  74,  622 

bottom,  62,  73 

of  bread,  564 

in  canned  foods,  528 

cell  of,  62 

classification  of,  61 

compressed,  559 

culture  of,  76 

cytology  of,  63 
.  definition  of,  61 

differentiation  of,  78 

diseases  caused  by,  775 

distillery,  73 

enzymes,  proteolytic,  of,  215 

in  fermentation,  73 

as  food,  563 

on  foods,  518 

on  grapes,  605 

morphology  of,  61 

pseudo-,  75,  77 

thermal  death-point,  276 

top,  62,  73 

wild,  77 

of  wine,  74 
Yellow  fever,  875 

ZONOCERCUS  elegans,  917 
Zooglcea,  89 
Zygomycetes,  34 
Zygorrhynchus  vuilleminii,  360 
Zygosaccharomyces  chevalieri,  70 
Zygospore,  38 

in  mucor,  49 

in  protozoa,  131 
Zymase,  207,  208,  214. 


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Marshall,  C.E.  M3 

Microbiology.  1921 


LIBRARY 

UNIVERSITY  OF  CALIFORNIA 
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